Beyond Efficacy: Ensuring Safety in Peptide Therapeutics through Immunogenicity Assessment

ABSTRACT

Peptides are gaining remarkable popularity in clinical diagnosis and treatment due to their high selectivity and minimal side effects. Over 11% of all new pharmaceutical chemical entities authorised by the FDA between 2016 and 2024 were synthetically manufactured peptides. A critical factor that can potentially limit the efficacy and safety of peptide‐based therapeutics or biologics is immunogenicity, defined as an unintended or adverse immune response to a protein or peptide therapy. This response may be triggered by the peptide itself or by impurities in the production or formulation steps, leading to the production of antidrug antibodies (ADAs). To address this, current regulatory guidelines require the assessment of risks in market authorization applications, which include identifying drug impurity levels and immunogenicity. The development and critical evaluation of appropriate immunogenicity assays is therefore highly warranted. Such assays must consider the fine complexities of the immune response, as well as its variation within the human population. Moreover, immunogenicity testing is expected to remain a priority as the shift toward greener chemistries in peptide synthesis may require reassessment of novel impurities in peptide formulations.

Peptides are increasingly used in diagnosis and treatment due to their selectivity and low side effects. Over 11% of FDA‐approved drugs from 2016 to 2024 were synthetic peptides. However, immunogenicity—an adverse immune response—can limit their safety and efficacy. This may result from the peptide itself or impurities, triggering antidrug antibodies. Regulatory guidelines now require immunogenicity risk assessment. Developing robust immunogenicity assays reflecting immune complexity and population variability is crucial and remains a priority as greener synthesis methods are introduced.

Abbreviations

AA

amino acid

ANDA

abbreviated new drug application

API

active pharmaceutical ingredient

AE

adverse events

ADAs

antidrug antibodies

ADCs

antibody‐drug conjugates

APCs

antigen presenting cells

BLT

bone marrow‐liver‐thymus

CAGR

compound annual growth rate

DC

dendritic cells

EPA

Environmental Protection Agency's

EMA

European Medicines Agency

FACS

flow cytometry

HLA‐II

human leukocyte antigen class‐II

ICS

intracellular cytokine staining

KLH

keyhole limpet hemocyanin

MHC

major histocompatibility complex

MAPPs

MHC‐associated peptide proteomics

NCE

new chemical entities

PBMCs

peripheral blood mononuclear cells

rDNA

recombinant deoxyribonucleic acid

RLD

reference listed drug

SPPS, LPPS

solid and/or liquid phase peptide synthesis

FDA

US Food and Drug Administration

1. Introduction

Considerable advancements have been achieved in the integration of peptides in the clinical diagnosis and treatment of a wide range of disorders in the last few years due to their rather low adverse effects and high selectivity [1, 2]. The astounding effectiveness of these treatments in several challenging therapeutic areas is demonstrated by the growing number of licenced medications available in the last decade [3]. Anticancer, antibacterial and antiviral properties represent some of the targets of this developing peptide therapeutic arsenal [4, 5]. Among the 420 drugs that the US Food and Drug Administration (FDA) authorised between 2016 and 2024, about 11% of all new chemical entities (NCE) were peptides (48) (Figure 1). It is noteworthy to mention that 9 of the 55 NCEs that were authorised in 2023—or 16% of the total—are peptides (Table 1) [1]. Several therapeutic peptide contenders are also in preclinical (~600) and clinical testing (~200) to assess their efficacy and safety in the treatment of various diseases [2, 8].

FIGURE 1.

Percentage of different classes of novel therapeutics approved by the FDA in the span of 2016–2024 of which TIDES (peptides and oligonucleotides) attribute for 11.4% (48 out of 420 total drugs) [6, 7]. Antibody‐drug conjugates (ADCs).

TABLE 1.

Number of novel FDA‐approved therapeutics as a total, peptides drugs and percentage of peptides from total for years 2015–2024 (https://www.fda.gov/).

Year	Number of total FDA‐approved therapeutics	Number of peptide therapeutics	% of peptide approved therapeutics
2024	50	2	4
2023	55	9	16.3
2022	37	6	16.2
2021	51	10	19.6
2020	53	5	9.4
2019	48	5	10.4
2018	58	4	6.9
2017	46	6	13
2016	22	1	4.5
Total	420	48	11.4

However, the usefulness of such therapeutics may be constrained due to immunogenicity that may lead to the formation of antidrug antibodies (ADAs), thus considerably limiting their efficacy and safety. To address this, both the FDA and European Medicines Agency (EMA) have provided guidelines for the assessment of risks in market authorization applications, which include identifying drug purity levels and immunogenicity. Initially, computational (in silico) methods rely on linear peptide sequences binding to HLA allele pocket, influenced by AA size, charge and polarity at each site. In vitro tests such as T‐cell culture testing, DC maturation assays, DC: T‐cell coculture assays and MAPPs evaluate immune cell activation following exposure to biotherapeutic substances or isolated impurities. Complementary to in vitro methods, researchers are adopting in vivo animal models since they provide a full immune system alternative, such as new HLA transgenic or humanised mice, to assess the likelihood of an ADA response and tolerance breach.

Despite the benefits of this growing market, the impact of toxic waste produced during the peptide synthesis poses an increasing challenge. Green chemistry, defined as the design of chemical products and processes that reduce or eliminate the use or generation of hazardous substances (US Environmental Protection Agency), is arising as a viable solution. Green chemistry concepts are revolutionising the pharmaceutical business through the reduction of hazardous waste and the alignment of manufacturing processes with international efforts to address climate change.

2. Manufacturing Process of Peptide Therapeutics

Most of the peptide therapeutics production used to be carried out through recombinant deoxyribonucleic acid (rDNA) technologies [9], whilst more recently, solid and/or liquid phase peptide synthesis (SPPS, LPPS) have been shown to be a more feasible approach for scale‐up manufacturing of novel drugs [10]. When it comes to crude peptides, SPPS is less complex than the recombinant production method, which demands the removal of biological components including enzymes, nonrelated proteins, DNA and RNA fragments [11]. AA coupling and deprotection are combined in a single reactor by SPPS, which simplifies peptide synthesis and has paved the way for the development of automatic peptide synthesisers [5].

In the final step of the synthetic process, the main peptide compound undergoes polishing and purification to minimise impurities that may interact with host immune cells and trigger undesirable responses before FDA approval. Since the contaminants in the final SPPS product are mostly the result of incomplete or unwanted reactions during the synthesis process, the detection is straightforward, and subsequent purification is not overly challenging [12]. The most frequent peptide process‐ or product‐related impurities are those caused by synthetic or formulation process related instability (oxidations, reductions, truncations, elimination, deamination, deletion, racemization, as well as aggregation, precipitation, etc.) [9, 13]. Additionally, depending on the chosen solvent's polarity and viscosity, along with the peptide sequence, the produced impurities are depended on and affected by the synthetic conditions.

3. Immunogenicity Guidelines for Peptide Approval

For manufacturing businesses in the pharmaceutical field, peptide therapies are a key class of drugs that are highly profitable. With over $70 billion in global sales in 2019, peptide medicines make up a sizeable share of the pharmaceutical industry, and over the next 10 years, the global market for peptide therapeutics is expected to expand at a compound annual growth rate (CAGR) of 10% [5, 8, 14, 15]. However, the formation of ADAs, that can neutralise or alter the clearance of peptide therapeutic candidates, reduce their efficacy or compromise safety by interacting with endogenous targets or causing drug‐induced hypersensitivity events, which are significant barriers to the application of these therapeutics [16]. One major and urgent need from the pharmaceutical industry is to anticipate immunogenicity challenges by choosing the least immunogenic pharmaceutical among the peptide candidates during the early stages of drug development, given the risk that these drawbacks halt the clinical development of new products [17]. Additionally, different production methods can increase the different impurities or solvent residuals whose presence may mediate immunogenicity reactions. Merely relying, however, on purification techniques to eliminate impurities, is inadequate, as purification is not always achievable. The development of novel preclinical testing strategies is warranted. The FDA and EMA have outlined, therefore, guidelines containing a list of considerations in order to assist the risk assessment that should be included in the market authorization application [11, 18, 19, 20].

Variations in contaminants and impurities, especially those linked to peptides, might impact a peptide drug product's efficacy or safety in comparison to the reference listed drug (RLD). According to the FDA guidelines (FDA‐2017‐D‐5767) on immunogenicity risk, any novel impurity must not have T‐cell epitopes with a higher affinity for major histocompatibility complex (MHC) molecules, nor should the proposed synthetic peptide change the function of the innate immune system. To further demonstrate the lack of an elevated risk of immunogenicity potential in the therapeutic product produced (active pharmaceutical ingredient [API] vs. the RLD), functional testing (in vitro or in vivo) can be conducted [21]. In order for an abbreviated new drug application (ANDA) to be submitted for the review of a proposed generic synthetic peptide, the following parameters should apply: (1) The level of any impurity in the proposed generic synthetic peptide should be lower or equal with the RLD, (2) if any new impurities in the proposed generic are higher than 0.5% of the substrate is not acceptable, (3) new impurities between 0.1% and 0.5% should be identified, characterised and justified for not affecting the safety and efficacy, including comparative immunogenicity risk assays [18]. Additionally, the FDA suggests a multitiered testing strategy and outlines the development and evaluation of ADA assays for screening (identification of antibodies binding to the peptide product), confirmation (specificity of ADA), titration (magnitude of response) and neutralisation (analysing ADA for neutralising activity) [22].

Similar to the FDA regulations, EMA guidance includes new in silico, in vitro and in vivo models as examples of developing technology tools that should be continuously considered for their potential application for development or preliminary assessments of clinical immunogenicity risk. Cell‐mediated responses may be exhibited using in vitro tests utilising innate and adaptive immune cells. The interference of the therapeutic protein in the ADA tests needs to be taken into consideration in toxicity investigations, since samples often include larger amounts of therapeutic protein. Blood samples should be collected and kept for later assessments as necessary to help interpret the research results, when ADA testing is not included in the protocol. Validation of the employed assays is also necessary [19].

4. Immunogenicity Risk of Therapeutic Peptides

For the immune system, the most immunogenic molecules are proteins. Immune cells, T lymphocytes in particular, are highly specialised in recognising peptide antigens, usually of pathogen origin, in order to mount a protective immune response. Therapeutic proteins and peptides, either created by live cells naturally, by bioengineering or synthetically, may therefore have the potential to trigger immunogenic reactions. In the latter cases, however, these reactions may have a negative impact on clinical outcomes by raising the likelihood of adverse events (AE) and/or lowering drug effectiveness. Some of the AEs seen as a result of an unexpected immunological reaction include reactions at the injection site, allergies, anaphylaxis, hypersensitivity and autoimmunity. This unfavourable immune response results in the production of ADAs, which can impact a biotherapeutic's pharmacokinetics (altering the drug's clearance rate), pharmacodynamics, effectiveness and even safety [23, 24]. Neutralising ADAs can disrupt both the biologic product's functionality while simultaneously can cross‐react with endogenous proteins, which are natural homologs to the drug leading to autoimmune reactions [9]. The immunogenicity of therapeutic peptides may be subject to a variety of factors attributed to either the patient receiving the medication, the treatment and/or the product itself. Genetic background, prior immunity (antibody pool), immunological state and immunomodulating treatment are patient‐related factors that may predispose an individual to a certain type of immune response. The dose schedule and delivery method are treatment‐related parameters. The manufacturing method, formulation, modifications and stability qualities of a synthetic product are factors that also impact the probability of an immunological response [19].

Immune reactions to biotherapeutics constitute a cascade of immune cell activation events that result in the production of ADAs. Dendritic cells (DC), macrophages and B cells, that act as antigen presenting cells (APCs), internalise the biotherapeutic, process it into peptides (antigens) and then display the individual peptide antigens on their cell surface in complex with MHC molecules, known as human leukocyte antigen class‐II (HLA‐II) molecules in humans. In this configuration, T‐cell receptors (TCR) on T lymphocytes can interact and bind to this peptide‐HLA‐II complex, triggering an initial stimulatory signal for the T lymphocyte. Costimulatory signals are required for T lymphocyte activation, proliferation and cytokine production in response to an antigen [21, 25]. Following their activation, T lymphocytes can lead to the production of ADAs by supporting the activation of B lymphocytes that are responsible for antibody production. ADAs may lessen the effectiveness of the API by attaching to and inhibiting the therapeutic protein's active site or by altering the protein's structure. The risk of unintended immunogenicity is often reduced for therapeutic proteins and peptides with a strong structural resemblance to endogenous proteins and peptides as T lymphocytes responsive to self‐peptides are clonally deleted as part of a process known as central tolerance [26].

5. Monitoring Immunogenicity at Different Levels of the Activation Cascade

Testing immunogenicity of peptide medications is crucial to ensure safety and efficacy as impurities arising during the synthesis may provoke undesirable immune responses [9]. Various methods are being developed and can be employed to assess the potential immune response to peptide therapeutics (Figure 2). The first stage in this step‐by‐step approach for assessing immunogenicity risk is the use of immunoinformatic techniques, i.e., in silico tools to analyse the impurity sequences in terms of HLA‐binding and epitope prediction. This is often used in conjunction with in vitro and in vivo studies that either confirm or invalidate the possibility of immunogenicity in later stages of the investigation [9].

FIGURE 2.

Assessment of immunogenicity risk of biotherapeutic peptides at different stages of the activation pathway of adaptive immune responses through computational methods (in silico) and in vitro assays for analysis of PBMC including DC, T and B cell cultures. Created with BioRender.com.

5.1. In Silico Methods for Sequence Prediction

In silico approaches, which use computational methodologies and simulations, are critical in predicting and identifying possible immunogenicity in peptide pharmaceuticals. Immunoinformatic analysis is a simple, economical and highly specialised method [27]. For instance, the Immune Epitope Database (IEDB) provides resources for epitope prediction for both MHC I and MHC II including EpiMatrix (EpiVax), NetMHCpanII, NNAlign and many others [28, 29]. Initially, the target AA sequence of the peptide in question is converted into FASTA format in order for the sequence to be entered into the databases [27]. Some of the ways in silico tools contribute to assessing immunogenicity include:

• HLA binding prediction: Peptides are presented to T lymphocytes via HLA molecules. Target AA connect to pockets located inside the HLA molecule's binding groove to initiate the HLA–peptide interaction. The ability of peptide sequences to bind to different HLA alleles may be predicted using in silico methods. Understanding the potential interactions between peptides and HLA molecules helps assess the likelihood of T lymphocyte recognition and activation [9, 30]. Immunogenicity risk evaluation frequently focuses on class II HLA‐DR. Nine prominent alleles are usually regarded as being quite significant for defining ADA: DRB1*0101, *0301, *0401, *0701, *0801, *0901, *1101, *1301 and *1501, since collectively, these variants encompass the genetic heritage of roughly 95% of the global human population [31].

• T‐cell epitope prediction/presentation: In silico methods can predict T lymphocyte epitopes, which are specific regions of a peptide, in this instance peptide drug epitopes, recognised by T lymphocytes. This information is valuable in assessing the potential for T lymphocyte‐mediated immune responses by identifying which epitopes of the drug can elicit a reaction.

In silico computational techniques based on algorithms depend on the capacity of linear peptide sequences to bind to the HLA allele pocket after being processed into overlapping peptides of up to 15 AA. Protein segment binding efficiency to a particular HLA allele is determined by the combined effects of AA size, charge and polarity at each site [32]. While the formation of antibodies against a biotherapeutic is what most testing for immunogenicity relies on, the in silico methods enable to shift the focus on predicting possible T helper cell epitopes, which would theoretically aid to increasing ADA‐producing plasma cells following the maturation of B cells [3]. Nevertheless, in silico tools have limitations including the inability of predicting immunogenicity for short peptides (3 to 8 AA), which cannot be tested since a sequence of 9 AA or more, is necessary. Owing to certain characteristics, short peptides are gaining more and more interest recently in the fields of biology, chemistry and medicine (a peptide with 3 AA recently approved Gly‐Pro‐Glu) [33, 34]. There are a lot of benefits that small peptides offer over their longer counterparts. Specifically, low‐cost synthesis (small and large scale), ease of modification, high bioactivity, accessibility, absorbability, as well as high safety and low toxicity (because of their safe metabolites, AA) and low immunogenicity [34]. The lack of representative data from in silico approaches has to be addressed since small peptides are generally anticipated to be nonimmunogenic, but it is still unclear how they would function in vivo. Additionally, non‐natural AA, nucleic acids, post translational changes occurring during the synthesis of the peptide (e.g., methylation and acetylation) and modified AA (e.g., tryptophan to bromotryptophan) are not yet accounted for by in silico techniques; instead, these aspects are required to be investigated by in vitro cell‐based T lymphocyte activation experiments [3].

5.2. In Vitro Assays for Evaluation of T Lymphocyte Activation

Aside from using immunoinformatics, FDA guidelines suggest performing in vitro assays for instance, using naïve donor peripheral blood mononuclear cells (PBMCs) from whole blood for T lymphocyte culture testing, DC maturation assays, DC: T‐cell coculture assays or MHC‐associated peptide proteomics (MAPPs) [35, 36, 37]. These techniques include assessing the level of immune cell activation after being exposed to either the biotherapeutic compound in its entirety or to extracted impurities originating from it [3].

The most applied method in immunological laboratories is a T‐cell‐based assay first published by Wullner et al. A crucial factor to consider when conducting T‐cell assays is guaranteeing adequate HLA coverage of the intended target group. Experiments must be planned so that PBMCs are obtained from numerous donors for each HLA supertype [9]. To evaluate the possible reactivity, T lymphocytes from healthy, unexposed individuals are often selected. Since CD4⁺ T helper lymphocytes are critical for the activation of antibody producing B cells, T lymphocyte assays have been established to test whether peptide drugs may stimulate new T lymphocytes circulating in patients in vitro [38]. This gives yield to an estimate of the proportion of T lymphocytes in healthy donors that are likely to elicit a response to the peptide in question. In this assay, cultured cells from naive donors are treated with the target drug(s), which include individual API and impurity peptides, and positive control antigen such as a highly immunogenic heterologous protein (i.e., keyhole limpet hemocyanin, KLH) and negative control human serum albumin [36]. When it comes to quantifying and characterising T‐cell response, flow cytometry (FACS) and intracellular cytokine staining (ICS) are the most efficient methods since they allow examination of several parameters at once. Following labelling, the cells may be sorted and measured, and cell surface markers and ICS can be used to identify the phenotype of T cells that respond to the antigen [32, 39].

Preclinical immunogenicity has significantly advanced through in vitro techniques particularly MAPPS, which can map the T‐cell epitopes on biotherapeutics following their natural degradation and presentation. As mentioned, protein therapeutics are naturally processed by DCs, which then present the resulting peptides in complex with HLA‐class II molecules. The MAPPS assay aims to identify possible T lymphocyte epitopes originating from HLA‐DR‐associated peptides derived from the biotherapeutic, which will in turn be able to activate T lymphocytes leading to unwanted immune responses. Therefore, techniques for eliminating potential T‐cell epitopes by inducing targeted AA alterations can be used to limit immunogenicity reactions [40, 41].

5.3. In Vivo Methods for Immunogenicity Prediction

A comparative evaluation of the possibility of an ADA response and tolerance break of an endogenous protein is currently addressed by various researchers using in vivo animal models, such as novel HLA transgenic or humanised mice (xenograft from human immune system) [3]. In contrast to in vitro and in silico models, animal models offer a complete immune system, which is why they pose an appealing alternative [42]. When evaluating the potential immunogenicity of protein drugs in humans, traditional animal models—which are mostly utilised in safety and toxicological studies for small molecule drugs—are not however very useful [40]. This is particularly true for human proteins, which animals have not developed tolerance against. Mice, rats and nonhuman primates have different MHC from humans at the AA level, even though their functions may be equivalent. This variation influences which T‐cell epitopes may be displayed, which in turn influences the immunogenicity of the recombinant peptide drug [32]. To overcome that issue, several transgenic mice strains have been created expressing the most prevalent HLA‐A, HLA‐B and HLA‐DR molecules [42]. An investigation with insulin analogues demonstrated that the transgenic mouse model (expressing human proinsulin) may identify neo‐epitopes resulting from protein alterations and breakdown of tolerance. Another study discovered a correlation between a rise in the number of substitutions in different insulin analogues and the prevalence of anti‐insulin antibodies [43]. Even with the development of such sophisticated models, transgenic mice are not as effective in predicting the prevalence of immunogenicity or the therapeutic impact of antibody production. For instance, Fradkin et al. investigated whether an aggregate‐containing solution might elicit an antibody response using a comparable GH transgenic mice model, which demonstrated that the aggregate did not stimulate the production of antibodies [44]. Overall, transgenic mice with human HLA allotypes are useful to investigate if an immune response is HLA‐restricted [45].

A substitute for transgenic mice models is the use of humanised mice, which exhibit tolerance to human immunoglobulins as well as human MHC class II (HLA) expression. These models rely on immunodeficient (with no endogenous NK cells) NOD/SCID IL2rγ^null or Rag2^−/−/γc^−/− mice, in which human CD34⁺ stem cells (usually obtained from human umbilical vein or PBMCs) are engrafted to neonates to replace their immune systems [46]. Recently, Sung and colleagues used humanised bone marrow‐liver‐thymus (BLT) immune mice in an FDA research study for testing a chemically synthesised peptide medication (Figure 3). They analysed two of the identified impurities of salmon calcitonin (immunogenic in silico and in vitro), which are known to trigger ADA production in patients. Teriparatide was utilised as a negative control since it has little immunogenicity in patients. According to the FACS results, the number of cells generating class‐switched immune responses was elevated by salmon calcitonin in a dose‐dependent fashion. With the use of this model, researchers were able to examine peptide medication dosages and exposure routes that may elicit immune responses from pharmaceutical compounds that are anticipated to be immunogenic [47]. Nonhuman primates are also a popular method to assess the immunogenicity of protein medications, in conjunction with mouse models. Nevertheless, nonhuman primates' MHC molecules still differ greatly from those of humans, which makes them less suitable as immunogenicity models [40].

FIGURE 3.

Bone‐thymus‐liver humanization surgery of SCID mice. Mice that had undergone sublethal radiation (immunodeficient) were implanted with human foetal liver and thymus tissue inside the kidney capsule. They were also given intravenous injections of pure CD34⁺ stem cells that had been isolated from the human foetal liver via magnetic positive selection to yield humanised mice [47]. Created with BioRender.com.

Apart from preclinical testing in animal models, in vivo methodologies include clinical testing with human participants for more representative data on adverse effects resulting from proposed peptides. Ethical concerns limit the number of human clinical trials conducted for immunogenicity testing of peptide products [42]. Nonetheless, clinical trials in every phase provide global organisations the data they need to apply for monitoring approvals. Finding the lead molecule in the early stages of drug discovery and developing an efficient production process are essential for evaluating the new drug candidate's safety and effectiveness in clinical trials [48]. Etelcalcetide was used (three times/week) in a 52‐week, multicenter, single‐arm, open‐label extension (OLE) Phase III trial (NCT01785875) to evaluate the long‐term safety and effectiveness of treating secondary hyperthyroidism in hemodialysis patients. In line with other studies that lasted for a shorter period [49], their results identified consistent decreases in serum parathyroid hormone after etelcalcetide therapy for a year with a tolerable safety profile. Anti‐etelcalcetide binding antibodies were found to be already present in most patients at baseline (exposure to etelcalcetide did not increase the incidence of ADAs) [50]. For therapeutic peptides that have been authorised since the start of 2010, the length of time spent in clinical development has varied significantly. A study published by Lau et al. reported that a cohort of 12 peptides had mean development duration of 9.4 years. Peptides that underwent shorter clinical development periods were generally accepted for use in conditions which had well‐established clinical trial endpoints, such as multiple myeloma (carfilzomib), type II diabetes mellitus (dulaglutide and albiglutide) and secondary hyperparathyroidism (etelcalcetide) [4].

6. Future Objectives: Green Chemistry, Safety Standards and Life Cycle Assessment (LCA)

The US Environmental Protection Agency's (EPA) states that green chemistry is the development of chemical substances and procedures with the goal of minimising or completely stopping the production of dangerous chemical derivatives [51]. Green chemistry principles are transforming the pharmaceutical industry, offering a sustainable approach to the synthesis of peptide therapeutics and redefining the foundations of peptide manufacturing. As environmental concerns escalate and regulatory restrictions tighten, adopting green solvents has become a regulatory and ethical imperative. Green chemistry not only reduces hazardous waste but also ensures that the manufacturing processes are aligned with global efforts to combat climate change and minimise environmental harm. Reliable methods for synthesising peptide biotherapeutics have made it feasible to successfully produce structurally demanding compounds for use in medicinal applications [52, 53]. One major area of concern is the high solvent consumption, which is a result of the numerous chemical steps required to synthesise the desired sequence and the repeated washings. Multiple washing steps are required to remove leftover reagents from the media prior to moving on to the next phase in the SPPS process, since there is no purification between each chemical phase [54]. These lengthy resin washes consequently produce a significant quantity of toxic solvent waste [55]. Peptide manufacturing processes typically produce 3 to 15 t of waste/kg of yield, based on the size of the peptide that is generated [56]. Green chemistry was formerly considered an ethical approach; but, in today's world, it has become an indispensable need due to the urgency of containing climate change, safeguarding the environment from pollution, limiting waste and ensuring that clean energy and water are accessible for future generations [53].

From an environmental and financial standpoint, pharmaceutical researchers that produce fine chemicals, recognised early on the significance of more environmentally friendly procedures due to the growing challenges of cost and sustainability [53, 57]. Novel greener technologies and consumables can be used as alternatives for the synthesis of peptides as current methodologies rely on the use of polar aprotic solvents such as N,N‐dimethylformamide (DMF) or N‐methyl‐2‐pyrrolidone (NMP), which are nephrotoxic and reprotoxic, have negative environmental effects when present in waste, and cause reproductive and developmental toxicity. In that context, conventional solvents are expected to be phased out gradually and replaced with safer and more ecologically friendly solvent mixtures as defined by Environmental Health & Safety (EHS) assessment criteria (e.g., ethyl acetate [EtOAc], acetonitrile [ACN], dimethyl sulfoxide [DMSO] and N‐butylpyrrolidinone [NBP]) [53, 55, 58]. For example, nearly a year ago, on December 12, 2023, the European Commission issued a regulation imposing restrictions on DMF, a compound widely used in various industrial applications across the European Union [59].

However, the adoption of greener alternatives faces several challenges. Transitioning to green solvents often requires significant investment in new infrastructure and process optimization. Furthermore, as alternative environmentally friendly solvents have yet to be used in large‐scale synthesis, scaling these processes to industrial production levels requires addressing reproducibility concerns. Differences in solvent polarity and chemical properties may affect peptide yield, stability and overall purity, requiring tailored protocols for each peptide synthesis from the beginning. The shift toward green chemistry also raises critical questions about the safety and efficacy of peptides synthesised with new solvents. Impurities arising from these processes, either due to incomplete reactions or solvent interactions, can influence peptide stability, aggregation and immunogenicity. Unlike traditional solvents, green solvents might also introduce novel impurities or change peptide characteristics in unforeseen ways. Variations in solvent properties could affect the folding, aggregation or degradation of peptides not only during the synthetic processes but also during the storage, packaging and delivery of the therapeutics. Understanding these effects is crucial for ensuring therapeutic safety as it is unclear what new impurities may be produced during synthesis and how the recipient's immune systems may respond to them.

This greener approach is especially important for peptide drugs that are used for life‐long treatment (such as liraglutide for diabetes), where patients will experience long‐term toxicity and immunogenicity issues as their action may be cumulative. Even trace levels of residual solvents or by‐products could trigger adverse immune responses, particularly in long‐term therapies. Using safer reagents for peptide biomolecules and minimising residual contaminants and traces of hazardous solvents in the final product will prevent unwanted adverse effects following administration. The transition toward greener technologies in peptide synthesis has already demonstrated a reduction in hazardous waste and a decrease in the environmental footprint of pharmaceutical production. Although adopting alternative solvents and sustainable practices addresses toxicity and waste issues, these efforts represent only part of the sustainability challenge. A more comprehensive approach involves evaluating the entire environmental impact of peptide production. LCA offers a holistic framework to analyse and evaluate the environmental effects of peptide production examining every stage—from raw material extraction and synthesis to purification, distribution and disposal. LCA manages to identify resource consumption, energy use and waste generation that might not be evident through conventional green chemistry approaches alone. For example, while greener solvents can reduce toxicity, an LCA might reveal increased energy demands or waste production in other phases, thereby advocating for a more balanced strategy. This comprehensive analysis not only complements initiatives that reduce hazardous waste and solvent toxicity but also uncovers hidden environmental hotspots, enabling the optimization of manufacturing protocols. Recent studies, such as those by Satta et al. and Gharate et al., highlight how integrating LCA into pharmaceutical production can drive improvements both environmentally and economically [60, 61]. Ultimately, embedding LCA into the peptide manufacturing process not only enhances sustainable practices but also supports the development of safer, more efficient therapeutic products.

Moving forward, research in peptide therapeutics should integrate LCA methodologies with green chemistry strategies to develop manufacturing processes that are both environmentally and economically sustainable. This integrated approach will be crucial in guiding the next generation of peptide drug development, ensuring that innovations in therapeutic efficacy are matched by advances in environmental responsibility. Furthermore, immunogenicity assays must provide adequate information that is lacking in this field. To establish whether the therapeutic peptides produced with green solvents are safe to be distributed to the public, a series of immunogenicity investigations are required. The road ahead demands innovation, collaboration and a steadfast commitment to safeguarding both human health and the environment. Large‐scale manufacturing using green solvents demands comprehensive immunogenicity testing of the green synthesised peptides integrating collaboration among academia, industry and regulatory bodies.

7. Conclusion

Every year, an increasing number of novel drugs are approved by the FDA of which the ever‐growing market of peptide pharmaceuticals is anticipated to shape the future of medicine and clinical applications with vast economic advancements. FDA and EMA regulations for approval of new peptide medication encompass the elimination of potential impurities from synthetic compounds that could lead to adverse effects and activation of the immune system. To characterise the immunogenicity status of the medications in people who have never taken them before, computational methods and wet approaches like cell cultures or animal models are required. Combining state‐of‐the‐art synthesis methods with comprehensive treatment approaches will remain crucial for addressing pressing medical challenges and enhancing healthcare worldwide. That entails reducing hazardous waste and improving the sustainability of the pharmaceuticals by using ‘greener’ environmentally friendly solvents in alternative production techniques.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.