How peptides are made: solid-phase synthesis, impurities and what "pharmaceutical grade" means

Most peptides in research catalogs, and many peptide medicines, are built by chemistry rather than extracted from tissue; most peptides today are manufactured by solid-phase synthesis [3]. How that chemistry works explains which impurities appear on a test report, and why the same molecule can be a controlled drug substance in one setting and an unregulated research chemical in another. This guide describes the manufacturing route and its typical by-products. It does not evaluate suppliers. For what each laboratory test measures, see purity testing explained; for reading a report line by line, see how to read a certificate of analysis.

Solid-phase synthesis in brief

Solid-phase peptide synthesis (SPPS) was introduced by R. B. Merrifield in a 1963 paper that reported the synthesis of a tetrapeptide [1]. In SPPS the chain is assembled stepwise while attached to a polymer support, one protected amino acid at a time [2]. Fmoc chemistry is now the method of choice, and multiton production of therapeutic peptides has made high-quality Fmoc building blocks available at low cost [2]. The older Boc method is now generally reserved for specialist applications [2]. At the end of assembly, trifluoroacetic acid (TFA) is used to cleave the peptide from the support [5]. Chemists from several large drug companies have described the current state of the art as relying mainly on legacy technologies that use large amounts of highly hazardous reagents and solvents [4].

Why impurities are built into the process

Because SPPS is a stepwise process, errors made at each deprotection and coupling step compound over the course of a synthesis [2]. A review of impurities in peptide medicines groups the synthesis-related types as follows [3]:

• Deletion sequences, missing one or more amino acids, which the review links to inefficient Fmoc removal [3].
• Insertion sequences, carrying an extra residue, linked to excess amino acid reagent [3].
• Truncated sequences, chains that stopped growing early. One cause is acetic acid contaminating Fmoc building blocks, which cannot be detected by reversed-phase HPLC and caps the chain permanently [2].
• Diastereomers from racemization, in which an amino acid residue is converted to its mirror-image form during synthesis [3]. Cysteine and histidine are especially prone; in one data set, histidine racemization rose from 1% without preactivation to 7.8% after five minutes of preactivation [2].
• Aspartimide-related products, a persistent side reaction of Fmoc chemistry at aspartic acid residues [16].
• Protection adducts and side reactions, from side-chain protecting groups that were not fully removed or from reagents reacting with unprotected side chains [3].
• Oxidation products and dimeric to oligomeric impurities [3].
• Counterions such as trifluoroacetate, carried over from synthesis or later purification [3].

The same review also found peptide products contaminated with unrelated peptides, which the authors attribute to a lack of appropriate GMP [3]. A second group of impurities forms after synthesis by degradation, including diketopiperazine, pyroglutamate and succinimide formation [3]. Storage-related change is covered in peptide stability basics.

Aggregation

Aggregation matters both during manufacture and in the finished material. On the resin, aggregation caused by association between growing chains is increasingly recognized as the major obstacle to synthesizing long peptides [2]. For finished generic peptide products, FDA's 2021 guidance asks applicants to show that a new impurity does not increase aggregation propensity or change the nature of the aggregates formed [7]. FDA cites the potential for aggregation and peptide-related impurities when explaining why compounded drugs containing several research peptides may pose a risk of immunogenicity [8].

Purification and freeze-drying

Synthesis yields a crude mixture, which is then purified; TFA serves as the ion-pairing reagent during purification, which is why synthetic peptides are commonly obtained as TFA salts [5]. The counterion can be exchanged: in one study, TFA was replaced with chloride by freeze-drying from dilute hydrochloric acid, with no effect on peptide purity [5]. Lyophilization, or freeze-drying, removes water from a product after it has been frozen, and FDA lists the enhanced stability of a dry powder among its advantages [6]. In sterile drug manufacturing, FDA describes the bulk solution being sterilized through a 0.22 micron bacteria-retentive filter and filled and partially stoppered under aseptic conditions before it is loaded into the freeze-dryer [6]. Whether a vial of powder is sterile therefore depends on those earlier steps, not on the drying itself [6].

How regulated manufacture sets impurity limits

FDA's guidance for generic versions of glucagon, liraglutide, nesiritide, teriparatide and teduglutide asks applicants to identify each peptide-related impurity present at 0.10% of the drug substance or greater [7]. It states that a proposed generic should not contain impurities at levels greater than those in the reference listed drug, and that a new specified peptide-related impurity should be no more than 0.5% of the drug substance [7]. The stated concern is immunogenicity: peptide-related impurities may create the potential for differences in immune response [7]. A regulatory assessor at the Italian Medicines Agency has argued that peptide-related impurities cannot be characterized with the approach used for small molecules, and that safety thresholds should follow specific risk identification [15].

cGMP versus "research grade"

Current good manufacturing practice (cGMP) for finished drugs is set out in 21 CFR parts 210 and 211, which contain the minimum methods, facilities and controls needed to assure that a drug has the identity, strength, quality and purity it is represented to possess [9]. Each batch of drug product must be tested for conformance to final specifications, including the identity and strength of each active ingredient, before release [10]. A drug made without complying with these regulations is deemed adulterated under section 501(a)(2)(B) of the FD&C Act [9, 11]. For the active ingredient itself, FDA guidance based on ICH Q7 covers GMP for manufacturing active pharmaceutical ingredients [12]. These documents define cGMP; none of them defines a "research grade" [9, 12]. In a 2026 warning letter, FDA stated that labeling peptides "research use only" and "not for human consumption" did not change its finding that the products were intended as drugs for human use [14]. The legal background is in peptide legality in the United States.

What "pharmaceutical grade" means

The phrase is tied to compendial standards. A pharmaceutical-grade compound is one for which a chemical purity standard has been established by a recognized pharmacopeia [13], and in US animal research the use of a non-pharmaceutical-grade substance must be scientifically justified when a pharmaceutical-grade version exists [17]. On that description the term refers to a compendial standard, not a purity percentage, so an unapproved peptide with no compendial monograph does not meet it however pure a test shows it to be [13]. The FD&C Act likewise measures a compendial drug against its compendium, treating it as adulterated if its strength differs from, or its quality or purity falls below, the compendial standard [11].

Questions to ask about how material was made

• Is manufacture described as cGMP, and if so, under which framework: 21 CFR 211 for finished drugs or ICH Q7 for active ingredients [9, 12]?
• Are related impurities reported individually, or only as a single total purity figure [7]?
• Is the salt form stated, for example TFA, acetate or chloride [5]?
• If "pharmaceutical grade" is used, is it tied to a compendial standard [13]?

Key points

• SPPS builds peptides stepwise, so small errors at each deprotection and coupling step accumulate [2].
• Typical by-products include deletion, insertion and truncated sequences, racemized forms, oxidation products, aggregates and residual counterions [2, 3].
• For generic peptide drugs, FDA asks that impurities at 0.10% or more be identified and that new ones stay at or below 0.5% [7].
• cGMP is defined in 21 CFR 210 and 211; "research grade" is not a regulatory category [9].
• "Pharmaceutical grade" refers to a compendial purity standard, not to a purity number [13].