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Process Mass Intensity (PMI): A Holistic Analysis of Current Peptide

Manufacturing Processes Informs Sustainability in Peptide Synthesis
Ivy Kekessie,* Katarzyna Wegner, Isamir Martinez, Michael E. Kopach, Timothy D. White,
Janine K. Tom, Martin N. Kenworthy, Fabrice Gallou, John Lopez, Stefan G. Koenig, Philippa R. Payne,
Stefan Eissler, Balasubramanian Arumugam, Changfeng Li, Subha Mukherjee, Albert Isidro-Llobet,
Olivier Ludemann-Hombourger, Paul Richardson, Jörg Kittelmann, Daniel Sejer Pedersen,
and Leendert J. van den Bos

Cite This: J. Org. Chem. 2024, 89, 4261−4282 Read Online

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ABSTRACT: Small molecule therapeutics represent the majority of the FDA-approved

drugs. Yet, many attractive targets are poorly tractable by small molecules, generating a need
for new therapeutic modalities. Due to their biocompatibility profile and structural versatility,
peptide-based therapeutics are a possible solution. Additionally, in the past two decades,
advances in peptide design, delivery, formulation, and devices have occurred, making
therapeutic peptides an attractive modality. However, peptide manufacturing is often limited
to solid-phase peptide synthesis (SPPS), liquid phase peptide synthesis (LPPS), and to a
lesser extent hybrid SPPS/LPPS, with SPPS emerging as a predominant platform technology
for peptide synthesis. SPPS involves the use of excess solvents and reagents which negatively
impact the environment, thus highlighting the need for newer technologies to reduce the
environmental footprint. Herein, fourteen American Chemical Society Green Chemistry
Institute Pharmaceutical Roundtable (ACS GCIPR) member companies with peptide-based
therapeutics in their portfolio have compiled Process Mass Intensity (PMI) metrics to help
inform the sustainability efforts in peptide synthesis. This includes PMI assessment on 40
synthetic peptide processes at various development stages in pharma, classified according to the development phase. This is the most
comprehensive assessment of synthetic peptide environmental metrics to date. The synthetic peptide manufacturing process was
divided into stages (synthesis, purification, isolation) to determine their respective PMI. On average, solid-phase peptide synthesis
(SPPS) (PMI ≈ 13,000) does not compare favorably with other modalities such as small molecules (PMI median 168−308) and
biopharmaceuticals (PMI ≈ 8300). Thus, the high PMI for peptide synthesis warrants more environmentally friendly processes in
peptide manufacturing.

P eptides have recently received a revived focus in the

pharmaceutical industry for reasons including their
biocompatibility profile, structural versatility, and potential to
explosive and sensitizing coupling agents,9,10 highly corrosive
trifluoroacetic acid (TFA), and other toxic solvents such as
dichloromethane (DCM), diethyl ether (DEE), and tert-butyl
modulate targets that are poorly tractable by small molecules.1,2 methyl ether (MTBE). These hazards are compounded by the
Solid-phase peptide synthesis (SPPS) has been a reliable large amount of solvent used for isolation and purification.
technology for the efficient synthesis of complex peptide targets, The Peptides Focus Team within the American Chemical
thus providing access to a broad chemical space in peptide Society Green Chemistry Institute Pharmaceutical Roundtable
therapeutics.3 However, SPPS and the vast majority of (ACS GCIPR) aims to facilitate a fast transition toward
technologies currently used in peptide synthesis require a large sustainable peptide synthesis. To achieve this goal, it would be
excess of hazardous reagents and solvents. Consequently,
pivotal to have accurate metrics for the current industrial
improvements are needed to reduce their environmental
impact.4−6 Problematic solvents include N,N-dimethylforma-
mide (DMF) and its close derivatives N,N-dimethylacetamide Received: July 4, 2023
(DMAc) and N-methyl-2-pyrrolidone (NMP). These solvents Revised: January 17, 2024
are globally classified as reprotoxic; their use may be restricted Accepted: March 1, 2024
and potentially banned in the near future.7,8 Other limitations of Published: March 20, 2024
peptide synthesis include poor atom-efficiency of fluorenylme-
thyloxycarbonyl protected amino acids (Fmoc-AAs), potentially
© 2024 The Authors. Published by
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4261 J. Org. Chem. 2024, 89, 4261−4282
The Journal of Organic Chemistry pubs.acs.org/joc Perspective

Figure 1. Select mass based metrics.

Figure 2. Illustration of solid vs liquid phase peptide synthesis cycles.

processes of peptide synthesis to identify where the greatest types of material and other factors such as energy usage, logistics,
impact can be made. This exercise has been extensively pursued environmental impact, or starting material complexity.
for small molecules, and the Oligonucleotide Focus Team of the Given the historical prominence of small molecules as
ACS GCIPR has recently conducted such exercise for medicines and their accessibility via well-controlled synthetic
oligonucleotides.11 However, there is a lack of industry-wide and analytical methods, it is not surprising that PMI was first
studies for peptides. We aim to bridge this significant gap in this developed for this modality. Typical small molecules have a
manuscript. reported PMI range of 168 to 308 kg material use per kg active
There are many established green chemistry metrics used to pharmaceutical ingredient (API).16 In contrast, large molecules
assess industrial processes.12,13 One of the most comprehensive (or “biologics”), which are biotechnology-derived molecules�
evaluations is a life cycle assessment (LCA) wherein the mass/ primarily monoclonal antibodies but also including fusion
energy balances and environmental impact of a product through proteins, traditional vaccines, etc.�have been found to have an
all stages (e.g., raw materials, production, end-of-life treatment, average PMI of ∼8,300 kg waste/kg API.17 Synthesized
disposal) is quantified. Since conducting a full LCA can be oligonucleotides, which are traditionally assembled in a
costly, time-consuming, and particularly challenging in early conceptually similar manner to synthetic peptides with an
phase development, simple mass-based metrics are routinely excess of reagents and solvents (as well as energy) via solid-
used (Figure 1). phase processes, challenging purifications, and burdensome
Atom economy (AE) quantifies the efficiency of a reaction by isolations, have been appraised within a PMI range of 3,035 to
measuring the number of reactant atoms that appear in the final 7,023 kg/kg with an average of 4,299 kg/kg.
product. It assumes both 100% yield and stoichiometric loading In this article, cross-company peptide PMI data for synthetic
and therefore provides a measure of only the reaction design. peptides will be analyzed using a methodology similar to that in
Conventional metrics such as chemical yield, conversion, and the oligonucleotides study. The unit operations are partitioned
selectivity quantify the actual conversion of the limiting reactant to better understand which stages are the most wasteful.11 In
to the desired product. However, like AE, they exclude other addition, evaluation of PMI per unit amino acids (AAs) for
significant resource requirements, such as solvent and other raw peptides could help shed light on the sustainability impact of
material inputs. peptides of different chain lengths.
Complete environmental factor (cEF), which is another
relevant metric to evaluate industrial processes, offers a measure
of the complete waste stream and also factors in all process
■ CURRENT PEPTIDE MANUFACTURING PROCESSES
Synthesis. As of 2020, greater than 100 peptide APIs have
materials such as solvents and raw materials.10,14 been approved for pharmaceutical use with sequence lengths
Furthermore, process mass intensity (PMI) provides a more ranging up to ∼60 amino acids.18 These include lengths ranging
holistic assessment of the mass requirements of a process, from approximately 5AA residues (e.g., thymopentin, 5AA) to
including synthesis, purification, and isolation. PMI is defined as more than 30AA residues (e.g., enfuvirtide, 36AA; teriparatide,
the total mass of materials used (raw materials, reactants, and 34AA; aprotinin, 58AA). Chemical assembly is typically carried
solvents) to produce a specified mass of product. It should, out via solid-phase peptide synthesis (SPPS) or liquid phase
however, be noted that PMI does not account for the peptide synthesis (LPPS) (Figure 2). While a linear sequence is
environmental impact incurred during the manufacture of typically generated by SPPS (including branching side-chains, if
starting building blocks and reagents. As PMI focuses on applicable), LPPS allows for both linear and convergent
maximizing value and efficiency, the ACS GCIPR has identified syntheses. Additionally, some specific LPPS-related methods
it as the key mass-related green chemistry metric and as an like soluble tag-assisted liquid phase peptide synthesis have been
indispensable indicator of the overall greenness of a process.15 A introduced.19−21 A hybrid approach, where SPPS synthesis of
limitation to the PMI metric is that it does not take into account short fragments followed by joining of the fragments via LPPS
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The Journal of Organic Chemistry pubs.acs.org/joc Perspective

has also been employed.22 Recombinant biotechnology or

enzymatic assembly of deprotected fragments is a further
orthogonal approach to chemical peptide synthesis.23
Regarding selection criteria for the synthesis strategy, aspects
to be considered include material demand, required batch size,
available manufacturing capability and capacity, complexity,
scalability, potential for automation, availability and costs of
starting materials, risk (process, timeline, costs), and regulatory
control strategy. The chosen approach should fit the API life
cycle as far as costs and timelines in development and
manufacturing are concerned, which may be subject to change
over time.
The LPPS strategy for the manufacture of shorter peptides,
containing 5−10 AAs for example, offers some advantages. This
approach allows for the utilization of different protecting groups
such as Boc and Cbz protected AA for the synthesis of complex Figure 3. Automatic 250 mL × 6 SPPS reactor.
targets. The need for step-specific process optimization in LPPS
provides the opportunity for limiting material and reagent usage,
reducing impurity formation, and increasing impurity rejection
during workup/isolation.24 In contrast to SPPS, the reaction
mixture may be analyzed by in-process analytical methods for
reaction monitoring. The LPPS strategy may be used with
conventional batch reactors; i.e., there are fewer restrictions with
manufacturing sites. However, LPPS does present a few
drawbacks. Typically, a greater effort is required to develop an
LPPS-based manufacturing process compared to SPPS, resulting
in higher initial costs and longer process development. Due to
the limited extent of automation, LPPS requires more manual
interactions during production, increasing risk due to human
error. Additional issues may include an increased risk of
racemization during coupling reactions and deprotections,
particularly during the peptide elongation process, which often
exhibits sluggish reaction kinetics. In prolonged reactions,
chemical instability issues may surface during the reaction and
Figure 4. Automatic 2 L × 4 SPPS reactor.
work up of the growing peptide chain, especially in cases of
larger peptides.
SPPS technology is a widely used and reliable platform.
Established building blocks, reliable supply chains, and well-
known chemistry conditions are available. Material generated
early in the API life cycle has likely been produced via SPPS,
demonstrating the general feasibility of the method, supported
by the availability of automated lab scale synthesizers. As a
consequence, less process development is required before scale
up. These aspects make SPPS an attractive synthetic strategy for
large-scale manufacturing of peptides. SPPS requires a filter
reactor, which typically is a specifically designed fully or
semiautomatic reactor (see examples in Figures 3, 4, 5, 6, and
7). At production scale, SPPS reactors with capacities from
hundreds of liters such as the automatic 250 mL × 6 SPPS
reactor (Figure 3) up to thousands of liters, as illustrated in the
6000 L SPPS reactor system (Figure 7), is employed.
Figure 5. Semiautomatic 100 L SPPS reactor.
Production-scale manufacturing of peptides with lengths in
the range of 70 AA via SPPS has proven feasible in principle.
However, even though the yield for each chemical trans- specific properties of a building block.27 In order to access more
formation is typically high, it is usually less than 100%. Hence, complex peptides or circumvent specific issues, alternative
despite the ambition to access ever larger peptides with varying approaches can be applied. One is the hybrid approach,28 which
chemical complexity by SPPS,26 the length of peptides that can combines linear SPPS of smaller peptide fragments with
be manufactured efficiently by SPPS has a practical limit of fragment condensation in solution, i.e. LPPS. Please note that
approximately 100 amino acids. Furthermore, when synthesiz- fragments may feasibly be manufactured by methods such as
ing longer peptides, sometimes specific issues like inefficient linear LPPS or soluble tag/anchor-assisted liquid phase peptide
coupling reactions, low yield, and excessive impurity formation synthesis as well, which would result in a convergent LPPS
may be observed, which are sometimes related to the formation process. An early example for production scale-manufacturing
of secondary structures of the protected peptide on the resin or according to the hybrid approach is enfurvatide,29 whereas a
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The Journal of Organic Chemistry pubs.acs.org/joc Perspective

or HPLC, and the characterization of the tagged growing

peptide is possible by NMR and MS analysis.
Chemical synthesis is the preferred method to produce
peptide therapeutics under development. Alternatively, re-
combinant or semirecombinant production is a valuable and
relatively sustainable strategy to produce peptides. The relatively
limited use of recombinant strategy is mainly driven by two
factors:31
(1) Currently established recombinant platforms can only
assemble peptides containing the natural L-AAs, limiting
the applicability of recombinant strategy. Research
activities are ongoing to extend the applicability of
recombinant strategies.32
(2) In general, the development time is significantly shorter
and the cost lower for the manufacture of peptides via a
Figure 6. 1000 L SPPS reactor.
chemical synthesis route.
Consequently, the application of recombinant strategies is
mainly focused on the large-scale manufacturing of long peptide
sequences comprised of natural L-AAs.23,33 A large-scale
recombinant synthesis of peptides has the advantage of
potentially driving cost down many folds. A key difference in
product registrations is that peptides produced recombinantly
are filed as Biologics License Applications (BLA), whereas
synthetic peptides are filed as New Drug Applications (NDA) if
their primary sequence is 40 amino acids or fewer. For synthetic
peptides with a sequence greater than 40 amino acids, they will
be filed as a BLA.
Purification. Purification of synthetic peptides is a major
challenge in the production of peptide APIs. The crude peptide
obtained by any of the aforementioned manufacturing strategies
Figure 7. 6000 L SPPS system. requires purification to achieve the specifications required for a
pharmaceutical API.
The number of iterative steps in peptide synthesis potentially
more recent example is tirzepatide.22 For the hybrid approach, generates a large number of impurities (e.g., deletion and
the peptide is subdivided into several fragments, which are truncation), with a chemical structure close to that of the target
separately prepared by SPPS and conjugated to the complete peptide. The separation of these impurities is therefore complex
peptide chain by LPPS in solution. The fragment approach adds based on the similarity of the chemical properties.
complexity and additional manufacturing steps compared to Crystallization can be a valuable option but with limited
linear SPPS, including the need for additional equipment. applicability to peptide manufacturing. It is mainly limited to
Nevertheless, the fragment approach may be more efficient than short peptide sequences or cyclic peptides and requires a high
linear SPPS; e.g., if a higher initial resin substitution may be initial crude purity as only a minimal increase in purity is
achieved, difficult coupling steps may be circumvented, resulting observed with longer sequences.
in a unique impurity profile that could make chromatography Purification Technologies. Purification is an integral part of
easier, a lower excess of AA derivatives may be sufficient, or a the synthetic peptide production process. Current processes for
higher purity and yield could be achieved. Moreover, the hybrid peptides are designed around chromatographic purification due
strategy has potential to reduce overall manufacture duration, to superior separation. Ion-exchange chromatography and
since the fragment production can be performed in parallel reversed-phase high performance chromatography are the
(convergent synthesis).30 most established techniques using differences in molecular
As both LPPS and SPPS have their specific limitations in charge and hydrophobicity, respectively.34
terms of complexity and environmental impact, modified LPPS The purification process impacts the resource consumption in
methods based on soluble tags/anchors have been proposed synthetic peptide production in two ways: First, the downstream
instead of an insoluble resin.19,21 With that technology, peptide processes account directly for approximately 50% of the Process
assembly takes place in a solution. The tag/anchor keeps the Mass Intensity (PMI), due to the use of dilute product solutions
growing peptide chain in the organic layer, which allows the and long chromatographic gradients. Although the created waste
removal of side products, excess building blocks, and coupling is rarely toxic and consists mainly of water, acetonitrile, and
reagents via aqueous extraction steps or precipitation. ethanol, organic solvents are not well-suited for recycling, as they
Consequently, less organic solvent than SPPS is employed. are strongly diluted in the process. Second, the purification
The technology is currently in use at multikilo scale for the process yield has a direct impact on overall PMI as the fully
manufacturing of short peptide sequences.21 As the tag is a synthesized product is handled, and losses in the downstream
relatively small molecule with a defined structure, both coupling process need to be offset by increased amounts in the upstream
and deprotection reactions could be monitored directly by TLC process, thereby increasing overall PMI.
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Reversed Phase, Ion Exchange, and Size Exclusion of separation principles, including ion-exchange, reversed-phase,
Chromatography. Preparative chromatography is the estab- and hydrophobic interactions.40
lished technology for the purification of most peptide APIs35 as Displacement chromatography can be very efficient, as no
it is known to be the most efficient approach to separate organic modifier is needed to form an elution gradient, and the
chemical substances with similar chemical structures. Large scale column can be loaded to maximum capacity. Additionally, the
high-pressure and low-pressure chromatographic units are used product elutes in a small volume from the column, reducing the
for the manufacture of peptides and are scalable on both lab- and need to concentrate and volume handling in subsequent process
production-scale. The purification of peptide APIs is typically steps. The significantly reduced solvent consumption makes
based on the use of reversed phase (RP) chromatography, ion displacement chromatography a considerable green alternative
exchange chromatography (IEC), and frequent size exclusion for synthetic peptide purification.
chromatography (SEC). Most of the manufacturing processes Supercritical Fluid Chromatography. Supercritical fluid
are based on an orthogonal strategy for purification (depending chromatography (SFC) uses liquified gases, often CO2, as the
on the impurity profile of the crude and its batch-to-batch liquid phase, which are enriched with additives to modulate
variability): 2 to 3 chromatographic steps are combined (RP peptide retention and peak shape. SFC is therein very efficient
and/or IEC). The proper strategy should target a complemen- due to the lower viscosity of the liquid phase and better diffusion
tarity between the purification steps to efficiently remove all properties which is providing a better separation when
impurities, based on the specific properties (e.g., pI, hydro- compared to classic RP-HPLC systems. This improved
phobicity).36 separation allows for much shorter gradients, thereby increasing
The most common solvent mixture used for peptide throughput and reducing solvent consumption.41 In pilot scale,
purification is water/acetonitrile. Alternatively, alcohols can be SFC can be performed with CO2 recycling,42 reducing the
used to replace acetonitrile. Ion pair additives/buffers can be environmental impact even further. The product is furthermore
included in the eluent to control pH, conductivity, and improve eluted in higher concentration, compared to RP-HPLC,
therefore reducing the need for subsequent solvent evapo-
separation. Gradient elution is often required except for very
ration.43
short peptide sequences.
The shifting focus to greener processes has sparked an
IEC can be used as an orthogonal method to RP, as it relies on
increasing interest in SFC; however, the high pressures and
different separation mechanisms. It can advantageously be used
dedicated equipment needed for SFC are limiting the
as a prepurification step to remove nonpeptide related application in larger scale for preparative separations.
hydrophobic impurities together with the residuals protecting Process Design. Mechanistic process modeling is an
groups and scavengers from cleavage. A final ion exchange step is important and well-established tool in the development of
often required after the last purification step to ensure the synthetic peptide purification process optimization strategies.
peptide is associated with the right counterion before going to Gétaz et al. showed that in silico models can be used to develop a
the final isolation step. multistep purification in production scale for a synthetic product
Preparative chromatography is by nature a highly dilute and gain significant advancement in yield.44
process, and the purification step is an important contributor to While the aim of optimization efforts is historically driven by
the PMI for peptide manufacturing. process yield and cost, reduction of the PMI is often not yet an
In the scope of greener purification processes, new develop- explicit optimization objective.
ments as well as established techniques can be regarded as For a truly efficient process regarding PMI, upstream and
suitable to decrease downstream PMI and to increase process downstream process steps need to be considered in the
yield. optimization efforts, given the impact of an increased yield in
Multicolumn Counter Current Solvent Gradient Purifica- late process steps on overall PMI. Vice versa, the potential of
tion (MCSGP). Multicolumn counter current solvent gradient process optimization in peptide synthesis might manifest in the
purification (MCSGP) uses multiple columns to retain impure overall process first in the reduction of purification steps or
fractions containing product from a primary column for increased yield in downstream processing. This shows that when
application on a secondary column and subsequent purification. optimizing processes, it is essential to assess the impact across
Initially developed with up to 8 columns, now systems with 2 the whole process and not only on the subprocess level,
columns are established in laboratory and production scale. especially in the synthesis-purification relationship.
While the process step mass index is not reduced directly, Isolation. The peptide API is generally isolated as a dried
increased yields compared to single column purification steps powder. The most common strategy is lyophilization (freezing
have been shown: Luca et al. showed for glucagon synthesized drying), which involves several steps. The dilute consolidated
on a solid phase that a yield increase of +23% could be achieved fractions contain the organic cosolvent and buffer used for the
on an optimized MCSGP process, compared to a single column elution of the peptide API. For industrial scale isolation, the
separation, while maintaining comparable purity profiles,37 pooled fractions are typically concentrated by removing the
thereby reducing the overall PMI. cosolvent before a final bioburden reducing microfiltration and
Displacement Chromatography. Displacement chromatog- freeze-drying. The concentration and cosolvent removal are
raphy uses the difference in binding strength of molecules to often performed using the following techniques:
achieve separation on a chromatographic column during • Tangential flow filtration with diafiltration of the solution
loading. Product elution can then be done with a displacement
agent which binds stronger to the column.38 Alternatively, in • Evaporation of the organic solvent
product displacement chromatography mode, multiple consec- Large scale tray lyophilizers can be used for the isolation of
utive columns are used which are then eluted separately, thereby API batches of less than 50 kg (Figure 8).
separating stronger binding impurities from the product.39 This Alternatively, the spray drying isolation technique offers great
mode of chromatography has been established for a wide range advantages for large scale applications with higher throughput
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Table 1. PMI Data for 14 Commercial and Phase 3 Peptides

Produced by SPPS
peptidea phase of development number of AAs PMI PMI per AA
X Commercial 8 18798 2350
M Commercial 10 19319 1932
L Commercial 8 10655 1332
BB Commercial 14 16671 1191
I Commercial 39 34585 887
CC Commercial 12 10370 864
E Commercial 19 11419 601
H Commercial 8 4414 552
A Commercial 7 3274 468
W Commercial 6 1683 281
R Phase 3 43 7008 163
U Phase 3 39 24742 634
Figure 8. Large scale tray lyophilizer (with associated isolator).
V Phase 3 15 12114 808
JJb Phase 3 43 7836 182
and lower energy consumption compared to freeze-drying. Average 19 13063 874.5
However, the peptide isolated product in this technique is a
exposed to high temperatures, which may negatively impact the Peptide sequences are blinded, and structures are not disclosed to
comply with proprietary policies of participating companies (Tables
purity and yield. This is often observed for the manufacturing of 1−4). bHybrid SPPS/LPPS.
batches where small quantities are processed.45
Assessing Sustainability of Current Processes. For this Table 2. PMI Data for 7 Phase 2 Peptides Produced by SPPS
article, 14 ACS GCIPR member companies have compiled PMI
metrics on 40 synthetic peptide processes at various develop- peptidea Number of AAs PMI PMI per AA
ment stages. These compounds included 34 SPPS processes, 4 F 30 15268 509
LPPS processes, 1 hybrid SPPS/LPPS, and 1 chemo enzymatic O 9 16299 1811
peptide synthesis (CEPS) process. Peptides were classified Q 9 19602 2178
according to phase of development, type of process, and number S 43 7205 168
of AAs in sequence. The synthetic peptide manufacturing Z 38 6504 171
process was divided into three stages (synthesis, purification, AA 12 10451 871
and isolation). The inputs for the entire process were summed to Average 23 12554 951
determine the PMI for each individual synthesis. For this a
Peptide sequences are blinded, and structures are not disclosed to
analysis, the route and PMI to Fmoc or Boc protected AAs were comply with proprietary policies of participating companies (Tables
not factored into the final PMI. These are typically considered 1−4).
readily available raw materials, and vendor information on PMI
is typically not available. It is acknowledged that the complexity Table 3. PMI Data for 14 Phase 1 or Preclinical Peptides
of the AAs will affect the mass efficiency of the overall process, Produced by SPPS
and future comparisons should strive to include this information
peptidea phase of development number of AAs PMI PMI per AA
to the extent that this is possible.
A summary of the output of this assessment is shown in Tables B Phase 1 34 79334 2333
1, 2, 3, and 4. The single hybrid SPPS/LPPS process was C Phase 1 15 27103 1807
grouped with the 34 SPPS processes since 90% of the waste D Phase 1 16 16277 1017
generating steps are from SPPS. Table 1 displays commercial G Phase 1 38 15889 418
and Phase 3 SPPS processes with PMI ranging from 1684 to J Phase 1 25 48306 1932
34,585 (average 13,063) for peptides ranging from 6 to 43 AAs K Phase 1 30 12617 421
in length (average 19 AAs). For better comparison between N Phase 1 9 38632 4293
peptide processes, PMI per AA was calculated in order to factor P Phase 1 9 22123 2458
sequence length into the PMI assessment. These values range T Phase 1 42 10337 246
from 163 to 2350 with an average of 874.5 for SPPS commercial DD Preclinical 17 14196 835
and Phase 3 processes (Table 1). The overall PMIs for synthetic EE Preclinical 36 74760 2077
peptides from SPPS were significantly higher than those for FF Preclinical 34 50309 1480
synthetic oligonucleotides. Synthetic oligonucleotides had an GG Preclinical 46 21296 463
average PMI of 4299 and average PMI per phosphoramidite HH Preclinical 46 32798 713
building block 199.11 The oligonucleotides process manufactur- Average 28 33141 1464
a
ing is mostly linear synthesis using established platform Peptide sequences are blinded, and structures are not disclosed to
methodology, whereas peptide manufacturing entails more comply with proprietary policies of participating companies (Tables
elaborate downstream operations with highly variable yield 1−4).
depending on the target structure (linear vs cyclic structure;
usage of natural amino acids vs. non-natural amino acids or small icals (PMI ∼ 8300).17 Limited innovation and improvements in
molecule building blocks) and its purity target. The SPPS PMI PMI are currently being observed at the later stages of
does not compare favorably either with other modalities such as development. This is depicted in the significant differences in
small molecule (PMI median 168−308)13 and biopharmaceut- the Phase 2 and 3/Commercial (Tables 1 and 2) vs Phase 1/
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Table 4. LPPS Processes

peptidea phase of development number of AAs PMI PMI per AA
A Phase 1 20 9019 451
B Phase 2 12 4280 357
C Phase 3 12 1139 95
D Commercial 10 1497 150
Average 13.5 3984 263
a
Peptide sequences are blinded, and structures are not disclosed to
comply with proprietary policies of participating companies (Tables
1−4).

preclinical processes (Table 3), with PMI per AA of 951/874.5

and 1464, respectively. A graphical representation of this
assessment is also depicted in Figure 9. The lack of PMI

Figure 10. PMI for LPPS vs SPPS.

and Phase 1, approximately double the waste is produced in the

purification steps, whereas the waste profile flips to higher
relative SPPS waste in the later stages. The trend may reflect the
use of oversized chromatography columns in the early clinical
phase and higher levels of washing required in a plant
environment for SPPS and/or not fully developed synthesis
conditions. Additionally, the low column loading, nonoptimized
chromatographic conditions, and poor crude peptide purity at
the initial process development contribute to high PMI at the
early development stage. Increasing purification PMI contribu-
tion in Phase 3/commercial compared with Phase 2 may be
related to a higher purity expectation of API for marketed
production. Isolation accounted for very limited use of solvent
Figure 9. Average PMI per AA for 34 SPPS processes per phase. primarily to just a single precipitation step for crude API and the
near exclusive use of lyophilization to deliver final purified
improvement in early phase development may be related to peptide API. While lyophilization is an energy intensive step
lower volume process manufacturing (<10 kg), limited process (energy factor is not included in PMI calculation), it is a
development, and aggressive time to start first in human studies. beneficial process for the isolation of the final peptide product
In addition, the lack of a downward trend in PMI as contributing to the PMI due to high yield, compared to
development progresses is inconsistent with what is generally alternative procedures such as precipitation, crystallization, and
observed with small molecule API.18 This may be because SPPS spray drying. Lyophilization is very efficient from a PMI
peptide development and manufacturing platform technology perspective as a standalone unit operation. Figures 11, 12, 13,
have not yet advanced to present opportunities where PMI can and 14 show that approximately equal amounts of solvents and
be reduced during the development lifecycle. An interesting water (44%) are used in SPPS processes. There is a similar
trend from the SPPS data sets is the increased peptide percentage of water used as in oligonucleotide processing
complexity in terms of manufacturing, often due to more (51%)11 but higher compared with small molecule processing
rigorous quality standards per phase of development (average (28%)15 due to the chromatographic purification steps. The
length commercial and Phase 3 = 19; Phase 2 = 23; Phase 1 = dominant organic solvent in SPPS is DMF (39%), which is the
28). However, the PMIs per AA for SPPS systems are static other main contributor to process waste due to extensive
across the development lifecycle, most likely due to the small washing of peptide on resin intermediates. Interestingly, the next
statistical differences in the number of AA on a manufacturing highest solvents used in synthetic steps, all at ∼1% individually,
scale. The most promising data submitted were from the 4 LPPS are MTBE, NMP, and 2-propanol. Acetonitrile (ACN ≈ 10%) is
processes, which account for 10% of the total processes the other ubiquitous waste that is used extensively in the
submitted (Table 4). The PMI per AA ranged from 95 to 451 chromatographic purification step.
(average of 263 per AA) for sequences 10−20 AAs in length. Safety Assessment vs Design Principles of Green
The performance differential between LPPS and SPPS processes Chemistry and Engineering. Safety is of paramount
is highlighted in Figure 10. This underlines the need to continue importance in any chemical enterprise and is at the heart of
to advance synthetic methodologies and technologies to sustainability principles. As outlined in the 12 principles of green
improve peptide therapeutic PMI across the development chemistry46 and engineering,47 several principles are grounded
phases. Interestingly, while the data set is limited, the decrease in safety (see Table 5, red). The peptide chemistry community
PMI across phase of development is consistent with PMI trends continues to identify hazardous reagents that are in current use
in small molecule commercialization. and offers guidance on their safe usage. Often significant
PMI was also subcategorized by unit operations (Synthesis, research and development is pursued to furnish alternative, safer
Purification, and Isolation) for SPPS processes. For preclinical reagents of equal or superior performance.
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Figure 11. Averages % of solvent usage of total PMI per process stage.

Hydroxybenzotriazoles are known for their shock and friction

sensitivity. Because dry 1-hydroxybentrotriazole is classified as a
class 1 explosive. The other hydroxybenzotriazoles, such as
HOAt, 6-Cl-HOBt, when dry also exhibit explosive properties.
For this reason, HOBt hydrate form is exclusively used.48 A
comprehensive list of 45 peptide coupling reagents has been
assessed for thermal stability, and a peptide reagent selection
guide has been developed from a process safety standpoint that
classified the reagents into “most preferred”, “use with caution”,
or “least preferred” categories.49 Ethyl cyanohydroxyiminoace-
tate (Oxyma) was deemed as a safer alternate to hydroxybenzo-
triazoles with lower risk of explosion based on calorimetry
studies (DSC and ARC).50 However, a recent study revealed
that HCN gas was detected under normal reaction conditions
Figure 12. Overall solvent composition SPPS process. during the use of Oxyma with DIC for AA activation.51 Since the
publication of this article, further efforts to mitigate the safety
risks of Oxyma have been reported. Usage of greener solvent
options and introduction of dimethyl trisulfide (DMTS) as a
HCN scavenger reduced the extent of HCN generation.52 A
recent study further highlighted the relationship of the
carbodiimide structure to the propensity to generate HCN,
finally offering safer alternates to DIC for peptide coupling.53
Peptide coupling reagents have been reported to be responsible
for severe allergic anaphylactic reaction.9,10 Several past reports
in allergy and medical publications were not picked up by the
synthetic chemistry community earlier. Since coupling reagents
can all modify human protein, there are risks of potential
sensitization, and therefore protocols for safe handling of
Figure 13. Solvent composition (synthesis). potential sensitizers must be followed.9 Global peptide
deprotection steps involved HF in the past when Boc- and Bn-
protected AAs were used during synthesis. A switch to Fmoc
synthesis was driven by safety concerns arising from the
handling of HF in the case of Boc peptide chemistry. In Fmoc
chemistry, TFA cocktails in organic solvents are used, typically
containing scavengers to avoid reincorporation of highly reactive
tertiary carbocations such as tBu often used for side chain
protection. Moving forward, alternatives to TFA are recom-
mended from a safety/sustainability standpoint.
In addition to safety considerations, many of the principles of
green chemistry and green engineering (Table 5, blue) aim to
maximize the mass and energy efficiency. Many of these
principles correspond to challenges in the sustainability of the
current approaches to peptide synthesis used today. The Fmoc
Figure 14. Solvent composition (purification). SPPS makes use of AA starting materials bearing Fmoc and side-
chain protecting groups. These starting materials are not aligned
with the “maximize atom economy” and “reducing derivatives”
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Table 5. Principles of Green Chemistry

Table 6. Summary of Solvent Composition for Synthesis and Purification Steps

synthesis step
Solvent DMF MTBE NMP IPA DIPE DCM Others ACN DMAC Heptane
% Norm 89.2 2.7 2.3 2.1 2 0.8 0.3 0.1 0.1 0.1
purification step
Solvent Water ACN EtOH MeOH IPA MTBE Acetic Acid
% Norm 77.3 18.4 2.2 0.7 0.7 0.5 0.3

principles. As noted above, most amide coupling conditions in that are advancing short-term peptide sustainability goals. Short-
peptide synthesis involve the use of benzotriazole derivatives as term goals are defined as areas that can be advanced in 10 years
stoichiometric reagents since viable and industrially applicable or less.
catalytic protocols are lacking. Finally, the goal of “waste Solvents. Solvents are the largest contributor to process mass
prevention”, which is linked to maximizing resource and mass intensity (PMI) and account for most of the waste generated and
efficiency, remains a significant challenge. Typically, peptide thereby the environmental impact of a drug substance. The PMI
syntheses require large excesses of stoichiometric reagents contribution of solvents is 80−90% for small molecules,55 and a
(Fmoc AAs, coupling additives, piperidine base) to ensure both high(er) percentage is applicable for peptides, especially
the coupling and Fmoc deblocking reaction proceed to considering the totality of solvents required during (a) solid
completion. Organic solvents and water constitute the greatest phase peptide synthesis, (b) solution phase chemistry such as
proportion of the waste generated as a large excess of solvent is cyclization, and (c) purification, usually by preparative reverse
routinely used to rinse the resin after each coupling and phase chromatography. DMF is the solvent of choice for SPPS
deprotection operation. coupling and Fmoc removal steps. Key reasons are favorable
Besides safety and mass energy efficiency, several other resin-swelling, ability to efficiently solubilize relevant starting
principles as outlined in Table 6 are relevant to the field of materials and reagents as well as byproducts, familiarity, and
peptide synthesis. Use of analytical technology such as in situ good knowledge around potential side-reactions with these
monitoring of Fmoc removal and the various washing steps solvents. However, DMF is a highly hazardous solvent, that was
(following coupling, capping, and Fmoc removal steps) can help restricted by EU REACH guidelines (December 2021) and was
in tailoring the solvent usage and avoid the waste of solvents and identified as SVHC (Substance of Very High Concern).56,57
reagents. Use of renewable feedstocks in the manufacture of key While water is the benchmark for green solvents, it is not fully
starting materials and reagents is a long-term sustainability goal. compatible with the existing SPPS technology. Recently,
Design for separation in the context of peptides can refer to any however, promising progress has been made toward the
advancement that can reduce the environmental impact of the development of water based SPPS where more water compatible
peptide purification step. Technology that helps in furnishing resins, Nα protected amino acids and reagents have been
cleaner crude peptide reduces the need for resource-intensive investigated.58 In the near term, finding a suitable replacement
chromatography.54 A comprehensive assessment of the for DMF/NMP that works with most common AAs and
sustainability challenges in peptide synthesis and purification reagents is critical to improve sustainability. Among the green
has been published that details best practices and areas of solvents studied, N-butylpyrrolidinone (NBP) was favored in a
improvement. study and was recently evaluated head-to-head against
Improving Sustainability in Near Term. The synthesis, DMF.8,57,59 NBP has been shown to exhibit superior perform-
purification, and isolation processes of peptide APIs have been ance compared to DMF in terms of reduced racemization and
greatly improved in the past few years since the GCIPR team’s aspartimide formation during SPPS. The high viscosity of NBP
last publication.5 These key processes of synthesis, purification, is a trade-off that results in inferior resin swelling and starting
and isolation, involving (i) solvent usage, (ii) coupling reagents, material solubilization compared to DMF. Also, the very high BP
(iii) new synthesis platforms such as solid tag-assisted LPPS, and (244 °C) would make it impractical from a solvent recyclability
(iv) nanofiltration/membrane-based reactor systems, are perspective. Alternate solvents exploration in SPPS spearheaded
discussed here as case studies on developments in the fields by the Albericio group includes ACN (Acetonitrile),60 THF
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(Tetrahydrofuran),60 MeTHF (2-Methyltetrahydrofuran),61 initiating the next AA coupling. Likely a reduction in piperidine
EtOAc (Ethyl acetate), 61 CPME (Cyclopentyl methyl concentration would impact the required solvent amount used
ether),61 GVL (γ-Valerolactone),62,63 NFM (N-Formylmorpho- for washing considerably and consequently reduce the overall
line),63 and PC (Propylene carbonate).6 Getting the right PMI of SPPS. Furthermore, Ravetti Duran et al. have
balance of properties to match the versatility of DMF has been demonstrated that applying a flow washing protocol after
challenging. GVL was found to react with AAs via ring Fmoc-removal (rather than batch washing) can reduce solvent
opening.64 While optimized conditions minimized this impurity consumption by more than 50%.71 A combination of reduced
during SPPS,62 there remains a continued need to investigate piperidine concentration and flow washing provides a simple
alternate organic solvents to afford chemists a broad solvent and ready-to-implement solution for reducing the PMI of SPPS
inventory to choose from based on their needs. considerably and deserves heightened attention by the industry.
Ideally, having a single solvent will simplify the execution of Coupling Agents and Recycling Reagents. Typically, with
the synthesis process. However, to strike a balance between the automation of SPPS, stoichiometric or excess reagents are
process friendly implementation and feasibility, binary solvent added and then washed away from the solid phase, leading to
mixtures have been considered as well. The advantage of having large amounts of waste being generated. One way to reduce this
a binary solvent pair is that the polarity can be tuned by adjusting waste burden is through a recycling strategy. Pawlas and
the solvent ratio according to solubility of reagents and Rasmussen have developed the concept of ReGreen SPPS, which
byproducts, since the Fmoc removal step is favored by polar not only focuses on optimizing the stoichiometry of the reagents
solvent mixtures, while the coupling steps are favored by a less in the various transformations but also on efficient recycling and
polar environment.2,65,66,57 Binary solvent mixtures were studied reuse of reagents/solvents thus fulfilling the criteria of a circular
wherein DMSO combined with either dioxolane (DOL), economy.72 To mitigate the issue regarding the use of
MeTHF, or EtOAc was of suitable high polarity for the Fmoc undesirable solvents in SPPS, the authors utilized mixed solvent
removal step. The coupling steps were favored in DOL with systems employing the common industrial solvent EtOAc as the
NFM or NBP. Another study on the binary solvent mixture bulk component. Initial results reported on an SPPS-protocol
considered c oligonucleotides yrene or sulfolane with diethyl using DMSO/EtOAc (1:9) on PS/DVB resin,73 which
carbonate (DEC), and anisole with DCM (dichloromethane).67 demonstrated improved chemoselectivity compared to conven-
Swelling of various resins was conducted, and a moderate tional solvent systems. This use of EtOAc as a cosolvent and its
dependence on the ratio of the binary solvent mixture was easy recyclability on industrial scale underpinned the develop-
observed. A subset of the best solvent combinations was chosen ment of ReGreen SPPS. Most notably, recycling of the waste
based on solubility of common Fmoc AAs and purity profile of
stream enabled 86% of EtOAc, 70% of DMSO, and 38% of
peptides synthesized using the green solvent pair vs standard
Oxyma to be recovered and reused in a new peptide synthesis
DMF. This approach is quite promising in the short term to
with minimum impact in compliance with regulatory require-
afford successful SPPS. In general, a marked dependence of the
ments. Benchmarking through life cycle analysis demonstrated
overall purity profile is associated with the solvent polarity, and
that although the cEF for the DMF and DMSO/EtOAc
hence, a change of solvent should be accompanied by careful
processes are comparable, implementation of the recycling
assessment of product purity and yield. Changing the solvent at a
very late phase of a project in a pharmaceutical setting could be into the latter (∼84% of the total waste) led to an ∼4-fold
challenging since new impurities in the latter batches might reduction in cEF with cutting of the solvent cost in-half per
necessitate additional toxicology and stability studies. It is amino-acid cycle (note further improvements of the latter are
recommended to consider sustainability aspects of the project envisioned through optimization of volumes used in the washing
early on, with early engagement by the project teams around steps).
implementing alternate solvents. An alternative paradigm is presented with the so-called “tea-
Additionally, piperidine is ubiquitously used to deprotect the bag” strategy, in which the polystyrene-resin located in a small
Fmoc groups, which is problematic since piperidine falls under propylene-sealed packet constitutes a microreactor, and this is
DEA list I of controlled chemicals (United States Drug the entity that is added to the reagents (for the reaction) or the
Enforcement Administration). Few alternative bases such as solvents (for the washing steps). This concept was first
methylpiperidines, DBU, piperazine, 4-methylpiperazine, and developed by Houghten74 in applying parallelization to SPPS
morpholine have been explored in DMF. Side reactions such as and though initially demonstrated for the Boc/benzyl protecting
aspartimide formation must be monitored closely since this is a scheme for peptide synthesis has subsequently been extended to
base-mediated side- reaction. Also, aspartimide formation is the more common Fmoc/t-butyl based protecting groups. The
affected by polarity of the reaction mixture, as shown in the case advantage in terms of reducing solvent/reagent amounts is that
of attenuated aspartimide formation when anisole was used as a one reagent solution can be utilized for multiple simultaneous
solvent.68 A recent report highlights the use of pyrrolidine to reactions (for example, 20% piperidine in DMF for Fmoc
deprotect Fmoc group in conjunction with greener solvents.65,69 removal) or on a number of discrete occasions for coupling of a
The poor AA solubility in anisole is countered by including a specific AA. In addition, wash solutions can be employed in a
cosolvent such as DMSO. There remains a need for a continued similar manner. To illustrate the power of this approach,
analysis of alternate bases as well as greener solvent Guzmán and co-workers reported on the synthesis of 52
combinations. peptides with a wide amino-acid composition ranging between 7
Alternatively, in 2004 Zinieris et al. published a study to 20 amino-acid residues.75 Though no correlation could be
investigating if the piperidine concentration could be reduced established between yield and physicochemical properties, the
for Fmoc-removal.70 No significant difference in SPPS perform- process showed excellent performance with a reduction in both
ance was observed between 5%, 10%, and 20% piperidine in DMF usage of 25% and a deprotection reagent of 50%.
DMF. After completed Fmoc-removal large amounts of solvent Recycling of reagents was integrated into the procedure with no
are used to remove byproducts and excess piperidine prior to adverse effect on the quality of the products generated.7
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Alternatively, Mainkar and co-workers have developed

benzoisothiazolone (BIT) as a fast, efficient, and fully recyclable
redox reagent that can be utilized for SPPS (Figure 15).77 Initial

Figure 16. Soluble tags for LPPS. (A) Phosphonate tag where R = Cl,
Figure 15. BIT recyclable reagent for SPP. OH, and N-OH for DDK-Cl, DDK-OH, and DDK-NOH respectively.
(B) Phosphonate tag where R = Cl, OH, and N-OH for TBP-Cl, TBP-
OH, and TBP-NOH respectively and R1 = H or Cl. (C) Silylated tag
proof of concept demonstrated that BIT-derived thioesters B2STag. (D) Silylated tag B6-STag.
could be formed through triethyl phosphite (P(OEt)3)-
mediated condensation of BIT with Fmoc-protected amino
acids and could be successfully condensed with a solid- stoichiometric equivalents of reagents (1.05−1.5 equiv) and
supported free amine in the presence of a Cu catalyst to recycle achieving >93% crude purity of the tagged peptide. The final
the BIT. The protocol was subsequently simplified with in situ peptide was still purified by RP-HPLC, but the overall reduction
formation of the thioester for coupling, which was demonstrated in chromatographic purifications and solvent usage is a large
and benchmarked favorably against conventional hexafluor- improvement over the previous demonstrations. This larger
ophosphate azabenzotriazole tetramethyl uronium (HATU) scale synthesis is a step in the right direction toward
and (EDC) 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimid implementing this technology for larger scale production.
based SPPS reactions with near stoichiometric quantities of the Yamagami et al.82 have also applied the tag-assisted
Fmoc-protected acid being utilized. Although 3 equiv of BIT technology to the synthesis of macrocycles, a modality with
were shown to be optimal, this reagent was recovered in 87% great therapeutic potential. Three different tagging positions
yield after a high-yielding gram-scale reaction. To highlight the were explored to form the macrocycle on the soluble tag, where
potential synthetic utility of this approach, SPPS of the the tag was either on the C-terminal, N-terminal, or in the
teixobactin side chain was demonstrated using BIT as a middle of the peptide. When the tag was positioned in the center
recyclable acid activator.78 of the peptide, the highest yield was achieved with no major
Greener Tag-Assisted LPPS (Tag LPPS). Tag-assisted liquid byproducts and without detectable epimerization. The authors
phase peptide synthesis (Tag-LPPS) has been further developed mentioned how the physical properties of the precipitate are
by research groups, as there were practical challenges that important for a successful process and that the tagging position
remained for effective scale up. Soluble supports have been may contribute to those properties, where the tag positioned in
expanded to include diphenylphosphonyloxyl diphenyl ketone the center provided precipitate with the best filtration
(DDK) tags, silylated tags, based on Chiba et al.’s lipophilic tags, properties. To effectively scale up this technology, it will be
and phosphonate tags, where both the silylated and important to better understand how to control the physical
phosphonate tags have a higher loading capacity than previously properties of the macrocycle precipitate and what variables can
developed LPPS supports (DDK tag;79 TBP tag;80 Silyl tag81) be optimized.
(Figure 16). Another advantage of the small molecule DDK and With these recent advances, the implementation of tag-
phosphonate tags is that the tag can be regenerated and recycled assisted LPPS is being realized. Bachem is working with Jitsubo
for reuse. These have been demonstrated on smaller scale but to use their Molecular Hiving technology, which employs these
are potential alternatives to advance tag-assisted LPPS. The lipophilic tag anchors, for larger scale peptide production (15−
lipophilic tags developed by Chiba and co-workers have 20 kg).83 Continued application of this technology to more
substantially progressed with application to macrocycles and a peptide targets of different sequences and modifications will be
larger scale demonstration in the last couple of years. Previous important to progress the method for broader applicability and
accounts still encountered long cycle times due to the required implementation on the manufacturing scale.
chromatography between each coupling step. To address the Organic Nanofiltration/Membrane-Based Reactor Sys-
lengthy processing time, Okada et al.21 utilized the hydrophobic tems. Organic nanofiltration and membrane-enhance peptide
benzylic alcohol (HBA) tags previously developed by their synthesis (MEPS) have been gaining traction in the field as a way
group, for larger scale peptide production. Icatibant acetate, a to purify growing peptide chains, remove chromatographic
Bradykinin receptor antagonist, was targeted and synthesized on isolations, and reduce solvent waste.22 Yeo et al.84 published
100 g scale. The larger scale was achieved by developing a one- work on PEPSTAR, which stands for peptide synthesis via one-
pot coupling, deprotection, and precipitation process. Quench- pot nanostar sieving. The technology uses organic solvent
ing excess activated AA with propylamine allowed the nanofiltration to purify growing peptides during solution phase
subsequent deprotection step to proceed without the need for synthesis. MEPS has been previously demonstrated but not
precipitation. As a result, the number of precipitations and scaled up due to lower loading capacity and lack of a
acetonitrile usage were reduced by half, while still using monodisperse anchor. In this most recent report, a mono-
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disperse anchor or “nanostar” was developed with an aromatic chemistry perspective and has the potential to cause side
core, making it detectable by LC and amenable for in-process reactions resulting in impurities.
monitoring. Additionally, 3 peptide attachment sites are If an economically low molecular weight alternative to the
available to increase the peptide loading and improve mass Fmoc-protecting group could be identified, then a shift away
efficiency due to the lower molecular weight of the nanostar. To from Fmoc could be envisaged for new processes. However, it is
facilitate a one-pot AA coupling, the activated ester was important that a holistic approach is taken to ensure that the
quenched, similar to the tag-assisted strategy, with excess problem is not merely removed from the SPPS stage, but rather a
piperidine used in the synthesis. This method was demonstrated negative environmental impact is achieved in the manufacture of
where the synthesis and purification stages use the same the required AA. Also, one has to ensure that the byproducts
equipment, resulting in a small footprint. Synthesis of di-, octa-, resulting from the deprotection of the N-α do not introduce new
and decapeptides was successfully executed with this automated environmental hazards. Provided that the environmental and
system and demonstrated the potential for larger scale financial incentive is clear, a shift to an alternative protecting
automation and production. The PMI was calculated and used group strategy could be envisaged in the mid- to long-term.
as a benchmark to optimize other LPPS technologies. The Peptide Bond Formation. Coupling reagents are highly
reported PMI is still close to that of solid phase synthesis, but the efficient at forming peptide bonds while ensuring that
authors anticipate that the PMI will decrease with inclusion of racemization and other side reactions are suppressed. However,
downstream operations. The cost of goods for the PEPSTAR excess reagents are required to drive reactions to completion. If a
technology was determined to be about half of that needed for catalytic peptide bond forming reaction was developed, this
traditional solid phase synthesis, making a compelling case for could reduce both the PMI and the use of toxic chemicals.
this technology. With the enhancements described, PEPSTAR Recombinant and enzymatic technologies already exist that
technology is a promising new technology for liquid phase synthesize peptides catalytically.87 The major drawback of these
synthesis that can be automated and potentially scaled up for platforms is that the current technology does not allow routine
larger production with continued progress. incorporation of unnatural AAs. The development of a
Improving Sustainability in the Long Term. The Fmoc- recombinant technology that allows the incorporation of
Protecting Group. The N-α Fmoc-protecting group and the side selected unnatural AAs could be envisaged. However, the real
chain protecting groups all add to the poor atom economy of landmark achievement would be the development of a
SPPS. As an example, the molecular weight of glycine is promiscuous system that would allow for the incorporation of
increased 4-fold after Fmoc-protection. If the Fmoc-protecting any unnatural AA.
group could be replaced by a simpler lower molecular weight Chemical catalytic peptide bond formation is another avenue
protecting group, the atom economy and overall PMI for SPPS of research that could significantly impact SPPS efficiency.
could be improved significantly. While considerable research has Considerable attention has been devoted to the area of catalytic
been devoted to identifying alternative side chain protecting amide bond formation during the past 20 years,88 but to date no
groups,85 largely driven by a desire to suppress side reactions, such system that can replace standard SPPS coupling reagents
very little attention has been devoted to identifying an has been identified, and only few examples on a scale beyond the
alternative to the Fmoc-protecting group. Imine based research laboratory have been reported.89
protecting groups based on dimethylbarbituric acid (DMB) Arora and co-workers have reported progress toward this goal
and diethylthiobarbituric acid (DETB)86 are two notable with the rational design of a biomimetic macrocyclic diselenide-
exceptions (Figure 17). The DMB group in particular has based organocatalyst. The catalyst operates through activation
shown promise as a replacement for the Fmoc-group; however, of the carboxylic acid component in the coupling as a selenoester
switching from Fmoc to DMB only results in ca. 25% reduction that undergoes the subsequent desired amidation reaction
in molecular weight. Furthermore, hydrazine is employed for (Figure 18).90 Critical to the success of the reaction is the
deprotection, which is an undesirable reagent from a process addition of a phosphine to initiate the sequence through
reduction of the diselenide and formation of a selenophospho-
nium derivative to react with the carboxylic acid as well as

Figure 17. DMB and DETB protected amino acids are synthesized
from enamine precursors 1. Deprotection is achieved using hydrazine to Figure 18. Diselenide-based organocatalyzed amide bonds, where R =
give the amino acid/peptide product and a pyrazole byproduct. desired AA side chain and X = AA residue.

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addition of a dehydrating reagent. However, both of these 750-M surfactant (Figure 18, see example 1 below).97 The
present barriers to sustainable implementation of this method- promises come from the encouraging preparation of a
ology as the optimal phosphine P(Bu)3 are prone to oxidation, decapeptide in either a 5 + 5 or 8 + 2 strategy in 72% and
while separation of dehydrating reagents such as molecular 82% yield, respectively. Considering the plethora of options
sieves can prove challenging in solid-phase based protocols. The reported by both academia and from industry,98 one could
amidation reaction is successfully demonstrated for Fmoc AA predict that this opportunity will soon be exploited, whether
derivatives using either DMF or ACN as the solvent and then using this surfactant or others now in the literature.99 A
impressively applied to the solid-phase synthesis of a significant impact on kinetics is observed when using the
pentapeptide. Removal of the sieves was achieved through the surfactants such as PS-750-M enabling rapid reactions in the
use of buoyant force with two coupling cycles required for order of 10 to 30 min to reach completion (Figure 20, Example
extension of the chain beyond the third amino-acid residue. 2).100
Purity of the final pentapeptide benchmarks well with that
obtained using more conventional SPPS-based coupling
approaches, while the hydrogen-bonded-based catalysis appears
to be unaffected by the presence of multiple amide bonds in the
molecule.
Most catalytic amide bond forming methodologies exhibit a
poor substrate scope. There have been some reports on more
general methodologies for the synthesis of amides91 and
peptides,92 but unfortunately catalysis has been achieved at
the expense of sustainability and does not offer a better
alternative to current stoichiometric methods. Chiba and co-
workers have recently reported an interesting approach using
electrochemical peptide synthesis in a biphasic system.93 Anodic
oxidation of triphenylphosphine provides the coupling reagent
in the form of the triphenylphosphine radical cation, producing
triphenylphosphine oxide as a stoichiometric byproduct after
coupling. The methodology is still in its infancy, and there are Figure 20. Peptide synthesis in water using a surfactant.
numerous challenges to overcome to make it of interest in an
industrial setting, but it represents a step in the right direction by Even more promising in terms of reaction acceleration has
utilizing a simple, inexpensive, safe, and general coupling reagent been the hydrophobic polymer effect reported by Braje at
(Figure 19). Abbvie (Figure 21).100 A remarkable kinetic enhancement was

Figure 21. Amide bond formation in water with addition of HPMC.

Figure 19. Electrochemical peptide bond formation using triphenyl-

phosphine, where PG = protecting group. observed when using HPMC (hydroxypropyl methylcellulose),
which, by virtue of the hydrophobic effect, promoted amide
To move this field forward, scientists must think about bond formation within seconds with high selectivity and
sustainability, simplicity, and robustness and demonstrate that chemical compatibility. Preliminary data regarding epimeriza-
the methodology is applicable on a multigram scale. tion were also promising.
Mechanochemistry for peptide bond formation is another In all these cases, a significant impact on the environmental
area with potential sustainability benefits, by dramatic solvent footprint and even total carbon release can be envisioned once
use reduction in the manufacture of small peptides.94 technical challenges associated with the contamination of the
The key next steps in the evolution of this field will be wastewater have been properly addressed.101
continued refinement of the approach, including description of
scope and limitations and demonstration of this approach on
scale-up.95,96
■ CONCLUSION
The ACS GCIPR has identified PMI as the key mass-related
A number of significant developments for peptide synthesis in green chemistry metric, which is a key indicator of sustainability
water enabled by surfactants has positioned water to serve as a for the manufacturing process of APIs. Although LCA and AE
potential bulk medium in an iterative process and a real long- are also alternative metrics in measuring the efficiency and thus
term alternative to reprotoxic polar aprotic solvents. Lipshutz for greenness of the process, LCA remains a challenge in early phase
example demonstrated a simple two-stage unmasking-coupling development, and AE excludes other significant resource
process that could be rendered amenable to peptide synthesis. requirements such as solvent and other raw material inputs.
Cbz can be smoothly removed from an AA with minimal amount Notwithstanding the noted limitation that PMI does not
of palladium on charcoal as a catalyst and hydrogen gas as the account for the environmental impact of starting materials
reductant, and subsequently engaged into the required amide (protected amino acid monomers), PMI still emerges as a
coupling step with very high efficiency, all in water with TPGS- preferred metric in this assessment.
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The current manufacturing processes for peptide APIs consist Philippa R. Payne − Outsourced Manufacturing,
of synthesis, purification, and isolation stages, with each stage Pharmaceutical Development & Manufacturing, Gilead
contributing to PMI. Data presented and discussed in this Alberta ULC, Edmonton T6S 1A1, Canada
perspective by 14 companies with peptide therapeutics in their Stefan Eissler − Bachem AG, 4416 Bubendorf, Switzerland
portfolio have underscored the persistent issue of high PMI for Balasubramanian Arumugam − Chemical Macromolecule
the manufacturing of peptides as therapeutics in comparison to Division, Asymchem Life Science (Tianjin) Co., Ltd., TEDA
that of small molecules. Solvent usage during synthesis and Tianjin 300457, China
purification contributes significantly to the PMI. From our Changfeng Li − Chemical Macromolecule Division, Asymchem
findings, PMI values for synthesis and purification range from 30 Life Science (Tianjin) Co., Ltd., TEDA Tianjin 300457,
to 70% of the overall PMI across development phases (Figure China
10). Additionally, SPPS and LPPS current strategies often Subha Mukherjee − Chemical Process Development, Bristol
employed in peptide synthesis have average PMI values trending Myers Squibb, New Brunswick, New Jersey 08903, United
more favorably for LPPS (Figure 9), although SPPS is the most States; orcid.org/0000-0002-9359-990X
widely used synthetic process for peptide manufacturing. The Albert Isidro-Llobet − GSK, Stevenage, Hertfordshire SG1
rather high average PMI value of 13,603 for commercial and 2NY, U.K.; orcid.org/0000-0001-9108-5916
Olivier Ludemann-Hombourger − PolyPeptide Laboratories
Phase 3 processes indicates the need for investigating and
France SAS, 67100 Strasbourg, France
advancing synthetic methodology and technologies to improve
Paul Richardson − Chemistry, Pfizer, San Diego, California
peptide therapeutic PMI across the production phases.102
09121, United States; orcid.org/0000-0002-3700-8749

■ ASSOCIATED CONTENT
Data Availability Statement
Jörg Kittelmann − CMC API Development, Novo Allé, 2880
Bagsværd, Denmark
Daniel Sejer Pedersen − Chemical Development, Novo Allé,
The data underlying this study are available in the published CMC API Development, 2880 Bagsværd, Denmark;
orcid.org/0000-0003-3926-7047
article.
Leendert J. van den Bos − EnzyTag B.V, 6361HK Nuth,

■ AUTHOR INFORMATION
Corresponding Author
Netherlands
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.3c01494
Ivy Kekessie − Early Discovery Biochemistry - Peptide
Therapeutics, Genentech, Inc., A Member of the Roche Group, Author Contributions
South San Francisco, California 94080, United States;
orcid.org/0009-0000-2844-9051; Email: Kekessie.Ivy@ The manuscript was written through the contributions of all
gene.com authors. All authors have given approval to the final version of
Authors the manuscript.
Katarzyna Wegner − Active Pharmaceutical Ingredient Notes
Development, Ipsen Manufacturing Ireland Ltd., Dublin 15,
The authors declare the following competing financial
Ireland
Isamir Martinez − Green Chemistry Institute, American interest(s): K.W. is an employee of IPSEN and owns stock or
Chemical Society, Washington, District of Columbia 20036,
stock options. M.N.K. is an employee of AstraZeneca and owns
United States
Michael E. Kopach − Synthetic Molecule Design and stock or stock options. S.M. is an employee of Bristol Myers
Development, Eli Lilly and Company, Indianapolis, Indiana
Squibb and owns stock or stock options. J.L. and F.G. are
46285, United States; orcid.org/0000-0002-3590-702X
Timothy D. White − Synthetic Molecule Design and employees of Novartis and own stock or stock options. L.J.vdB.
Development, Eli Lilly and Company, Indianapolis, Indiana
owns stock or stock options in EnzyTag. O.L.H. is an employee
46285, United States
Janine K. Tom − Drug Substance Technologies, Amgen, Inc., of PolyPeptide Laboratories. B.A. and C.F. Li are an employee of
Thousand Oaks, California 91320, United States;
Asymchem Life Science own stock or stock options. A.I-L. is an
orcid.org/0000-0002-3531-8751
Martin N. Kenworthy − Chemical Development, employee of GSK and owns stock or stock options. I.A.K. and
Pharmaceutical Technology & Development, Operations,
S.G.K. are employees of Genentech, a Member of the Roche
AstraZeneca, Macclesfield SK10 2NA, United Kingdom
Fabrice Gallou − Chemical & Analytical Development, Group, and own stock or stock options. M.E.K. is an employee of
Novartis Pharma AG, 4056 Basel, Switzerland; orcid.org/
Eli Lilly and Company. S.E. is an employee of Bachem and owns
0000-0001-8996-6079
John Lopez − Chemical & Analytical Development, Novartis stock or stock options. P.R. is an employee of Pfizer Inc. and
Pharma AG, 4056 Basel, Switzerland; orcid.org/0000-
owns stock or stock options. D.S.P. is an employee of Novo
0002-0551-6511
Stefan G. Koenig − Small Molecule Pharmaceutical Sciences, Nordisk and owns stock. J.K. is an employee of Novo Nordisk
Genentech, Inc., A Member of the Roche Group, South San
and owns stock. All other authors declare no competing financial
Francisco, California 94080, United States; orcid.org/
0000-0002-1878-614X interest.
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The Journal of Organic Chemistry pubs.acs.org/joc Perspective

Biographies ́
Isamir Martinez, Ph.D., is currently the Scientific Alliances and Business
Engagement Sr. Portfolio Manager at the ACS Green Chemistry
Institute/Office of Sustainability. Isamir is leading efforts to strategically
accelerate and enable the implementation of green and sustainable
chemistry and engineering throughout the global chemical enterprise.
In her role, she leads global industrial collaborations, engages
stakeholders, and other strategic collaborative programs that contribute
to sustainability. She has a wide array of extensive scientific background
in organic, medicinal/process/pharmaceutical chemistry, biocatalysis,
and chemical sourcing. Prior to joining ACS, she worked in the
pharmaceutical industry for over 12 years and taught chemistry at
higher education institutions. Isamir’s passion is to develop green and
sustainable chemistry methodologies that could be used to deliver
efficient chemical processes and therefore contribute to a more
sustainable future.

Ivy A Kekessie obtained her PhD in Organic Chemistry from the

University of California, Davis, CA under the supervision of Professor
Gervay-Hague with research focused on the design and synthesis of
HIV-1 entry inhibitors. Her postdoctoral studies with Professor Kit
Lam at the University of California Davis Medical Center, Sacramento,
CA, involved design, synthesis, scale up, and formulation of
nanomicelles as delivery vehicles for chemotherapeutics targeting
various cancer cells. Ivy joined Genentech (A Member of the Roche
Group) in South San Francisco, CA in 2015 where she is currently a
Scientist in the Peptide Therapeutics department working on peptide-
derived therapeutics.

Michael Kopach earned his Ph.D. in Organic Chemistry from the

University of Virginia in 1995. Following postdoctoral studies with
Prof. A. I. Meyers at Colorado State University in natural product
synthesis, he joined Roche in 1997. At Roche, he contributed to the
Development group’s efforts in commercializing AIDS drugs such as
Viracept and Fuzeon, the latter being the first synthetic peptide to reach
commercialization on a large scale. In 2001, Dr. Kopach became a part
of Eli Lilly and Company’s Synthetic Molecule Process Chemistry
Division. Over the past decade, he has led Eli Lilly and Company’s
initiatives in synthetic peptide commercialization and sustainability.
Notably, he was instrumental in the route design and commercial
implementation of Mounjaro, recognized as the first GLP-1 and GIP
Katarzyna Wegner obtained her PhD. in Organic Chemistry (2007), hormone dual agonist.
Faculty of Chemistry, University of Gdańsk under the supervision of
Professor Zbigniew Grzonka. In 2008, she joined IPSEN, Ireland where
she is currently the Director of Active Pharmaceutical Ingredient
Development group responsible for improvement and innovation of
novel peptide and small molecule process manufacturing.

Tim is a Senior Director at Eli Lilly and Company with responsibilities

for peptide and oligonucleotide process development activities. He has
25 years of experience as a process chemist with 9 years at Pfizer prior to
joining Eli Lilly. Tim has worked across small molecules, antibody drug
conjugates, peptides, and oligos and has a long-standing interest in

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improving sustainability and greening of chemical processes with large Fabrice is a Distinguished Scientist at Novartis, Switzerland,
scale manufacturability in mind. responsible for scientific activities in Technical Research & Develop-
ment. His research interests include the research and development of
sustainable synthetic methodologies intended for large scale
implementation.

Janine Tom obtained her Ph.D. in Chemistry from UC Irvine in the lab
of Professor Aaron Esser-Kahn, where she developed synthetic
chemical tools to probe the innate immune response for vaccine
development. She started at Amgen in 2017 and is currently a Process John is a senior expert in science and technology at Novartis. His main
Development Senior Principal Scientist in the Drug Substance responsibility is process development for the manufacture of peptides in
large scale, and additionally he is very interested in sustainability and
Technologies Synthetics group. At Amgen, she worked on several
innovation towards data science applied to organic chemistry.
synthetic and hybrid modality programs, including traditional small
molecule, antibody-drug conjugate, and siRNA modalities.

Stefan G. Koenig obtained his PhD in Organic Chemistry from Yale

University in New Haven, CT (USA), under the supervision of
Martin Kenworthy studied Chemistry at University of Manchester
Professor David J. Austin and then conducted postdoctoral work at the
(UK) and obtained his Ph.D. in 2003 with Prof. Jonathan Clayden. ETH in Zurich, Switzerland, in the laboratory of Professor Andrea T.
After postdoctoral studies with Prof. Richard Taylor at York University Vasella. Stefan initiated his professional career at Sepracor in Marlboro,
(UK), he joined AstraZeneca in 2006 (Macclesfield, UK), where he MA, and joined Genentech (A Member of the Roche Group) in South
works in Chemical Development. Over the past decade, he has been San Francisco, CA in 2010. He is currently a Distinguished Scientist in
responsible for AZ drug substance development for late clinical stage the External Science & Manufacturing group within Synthetic Molecule
and commercial peptide projects and products. Pharmaceutical Sciences, leading various projects at partner companies.

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The Journal of Organic Chemistry pubs.acs.org/joc Perspective

Philippa (Pippa) Payne is an Associate Director in Global external

manufacturing at Gilead Sciences. She completed her Ph.D. at the
University of British Columbia, followed by postdoctoral studies at the
University of North Carolina − Chapel Hill. At Gilead Sciences, she has
worked on late-stage and commercial small molecule and peptide
programs, advancing the application of green chemistry principles in
process development and manufacturing. She was an elected cochair of
the ACS Green Chemistry Institute Pharmaceutical Roundtable from
2021−2023.

Changfeng Li obtained a Ph.D. in Organic Chemistry from Central

China Normal University at Wuhan of China in 2009. He is currently
working as a Senior Director of peptide team at CMMD, Asymchem.
He is responsible for the CDMO business for SPPS and polypeptide
laboratory management. Responsibilities also include customer
management, process development, and production project manage-
ment of early phase projects.

Stefan Eissler studied biochemistry at Bielefeld University and

completed these studies with a PhD thesis in organic chemistry
under the supervision of Norbert Sewald. After a postdoc position at the
laboratory for process research at the university of Zürich under the
supervision of Thomas Bader, he joined Bachem AG in 2010 as a team
leader. After various positions as team leader and director responsible
for liquid-phase and solid-phase peptide synthesis as well as peptide
purification, he currently holds the position of the Vice President Subha Mukherjee earned his PhD in Chemistry from the University of
Illinois at Urbana−Champaign in 2015. He worked under Professor
Peptide Manufacturing Upstream. Wilfred A. van der Donk’s supervision in peptide synthesis, bio-
orthogonal chemistry, and chemical and enzymatic synthesis to
investigate lanthipeptide and phosphonate natural products. In 2015,
he joined the process chemistry department at Bristol Myers Squibb
Company. Over the years at BMS, he has transitioned from enabling
scalable process development to leading chemistry teams in the peptide,
small molecule, and ADC portfolio as a modality-agnostic process
chemist.

Arumugam Balasubramanian (Bala) earned a Ph.D. in Chemistry from

National Tsinghua University Taiwan, currently working as Director of
research at Asymchem Tianjin for the process development and
manufacturing of oligonucleotides, peptides, and small molecules for
early to late phase molecules. He has experience in peptides and
oligonucleotides process development and manufacturing with Albert Isidro-Llobet obtained his PhD in Organic Chemistry (2008) at
expertise in synthesis, purification, and isolation. the Institute for Research in Biomedicine, Barcelona Science Park,

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The Journal of Organic Chemistry pubs.acs.org/joc Perspective

University of Barcelona under the supervision of Professors Fernando

Albericio and Mercedes Alvarez. His PhD work involved the
development of methodologies for peptide synthesis. In 2008, he
received a Marie Curie Fellowship and joined the group of Prof. David
Spring at the University of Cambridge where he worked on the
diversity-oriented synthesis of macrocyclic peptidomimetics. He joined
GSK, Stevenage, UK in 2012 as a peptide synthesis expert. He currently
works at the Chemical Biology department in GSK where his role
involves leading peptide, target identification, and data analysis
projects.

Jörg Kittelmann is a Principal Scientist at Novo Nordisk specializing in

purification process development for peptides and proteins. He has over
8 years of experience at the company and is very passionate about
process sustainability and process analytical technologies. Jörg holds a
PhD in Biotechnology from Delft University of Technology.

Olivier Ludemann-Hombourger is the Global Director Innovation &

Technology at PolyPeptide. He received his PhD in 2001 from the
“Institut National PolyTechnique de Lorraine” in France, in
collaboration with Novasep Process, for which he was leading the
R&D activity over 10 years. He worked during three years for Lonza in
Daniel Sejer Pedersen obtained his PhD degree in synthetic organic
Braine l’Alleud (Belgium) as head of Operations, before joining chemistry from Cambridge University in 2005 and has held a variety of
PolyPeptide as General Manager of the French entity for 7 years, until positions at universities and in industry. He is currently a Specialist in
CMC Chemical Development at Novo Nordisk where he has been
taking his current global position in 2019. employed since 2018 working on synthetic phase 3 projects and the
assessment and development of new manufacturing technologies.

Paul Richardson earned his Ph.D. in Chemistry from the University of

Sheffield (Synthetic Methodology/Total Synthesis, Professor Istvan Dr. Leendert van den Bos obtained his MSc and PhD degree in bio-
organic chemistry from Leiden University. He joined Organon
Marko) and did postdoctoral studies at the University of Exeter
BioSciences as a Scientist in 2007 and worked subsequently for
(Biocatalysis, Professor Stan Roberts) and The Scripps Research Schering-Plough, Merck Sharp & Dohme and Aspen Pharmacare
Institute (Asymmetric Catalysis, Professor Barry Sharpless). He is mostly in the field of Process Research & Development. Leendert holds
an Executive MBA from Nyenrode University. His research interests
currently the Director of Analytical and Synthesis Methodologies include process development, enzyme engineering, and continuous
within Oncology Medicinal Chemistry at Pfizer in La Jolla. flow manufacturing.

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■ ACKNOWLEDGMENTS
This manuscript was developed with the support of the
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■ ABBREVIATIONS
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