JOC Peptides

Perspective
Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/joc
Sustainability Challenges in Peptide Synthesis and Purification:

From R&D to Production
Albert Isidro-Llobet,*,† Martin N. Kenworthy,*,‡ Subha Mukherjee,*,§ Michael E. Kopach,*,∥
Katarzyna Wegner,*,⊥ Fabrice Gallou,*,# Austin G. Smith,*,⊗ and Frank Roschangar*,∇
†
Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, U.K.
‡
Pharmaceutical Technology and Development, AstraZeneca, Silk Road Business Park, Charter Way, Macclesfield SK10 2NA, U.K.
§
Chemical and Synthetic Development, Bristol-Myers Squibb Company, One Squibb Drive, New Brunswick, New Jersey 08903,
United States
∥
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Small Molecule Design and Development, Eli Lilly and Company, 1400 West Raymond Street, Indianapolis, Indiana, United States
⊥
Active Pharmaceutical Ingredient Development, IPSEN Manufacturing Ireland, Ltd., Blanchardstown Industrial Park, Dublin 15,
Ireland
Downloaded via UNIV OF FLORIDA on March 22, 2019 at 14:22:49 (UTC).
#
Chemical & Analytical Development, Novartis, 4056 Basel, Switzerland
⊗
Drug Substance Process Development, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, California 91320, United States
∇
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877, United States
ABSTRACT: In recent years, there has been a growing interest in

therapeutic peptides within the pharmaceutical industry with more
than 50 peptide drugs on the market, approximately 170 in clinical
trials, and >200 in preclinical development. However, the current
state of the art in peptide synthesis involves primarily legacy
technologies with use of large amounts of highly hazardous reagents
and solvents and little focus on green chemistry and engineering. In
2016, the ACS Green Chemistry Institute Pharmaceutical Round-
table identified development of greener processes for peptide API as
a critical unmet need, and as a result, a new Roundtable team
formed to address this important area. The initial focus of this new
team is to highlight best practices in peptide synthesis and encourage much needed innovations. In this Perspective, we aim to
summarize the current challenges of peptide synthesis and purification in terms of sustainability, highlight possible solutions,
and encourage synergies between academia, the pharmaceutical industry, and contract research organizations/contract
manufacturing organizations.
T he American Chemical Society (ACS), the largest

scientific society in the world, hosts the ACS Green
Chemistry Institute (GCI) with the mission to catalyze and
weight range between 1000 and 5000 Da. Many peptide
products fit this definition and are an area of significant growth
within the biopharmaceutical industry with more than 50
enable the implementation of green and sustainable chemistry peptide drugs on the market, approximately 170 in clinical
and engineering throughout the global chemical enterprise and trials, and >200 in preclinical development.4 However, the
across the Society.1 To meet its focus to accelerate industrial current state of the art in peptide synthesis involves primarily
adoption of green chemistry, the ACS GCI launched a series of legacy technologies with use of large amounts of highly
industrial, noncompetitive collaborations (called Roundtables). hazardous reagents and solvents and little focus on green
In 2005, the ACS GCI Pharmaceutical Roundtable (GCIPR) chemistry and engineering, though pressure to utilize more
was formed with three companies, and membership now has benign substances is growing.5 For instance, significant
grown to 29 in 2019. portions of coproduced waste streams are the hazardous
In the early years, the Roundtable was focused exclusively on solvents N,N-dimethylformamide (DMF) and N- methyl-2-
small molecule chemistry but has gradually evolved to address pyrrolidone (NMP), used in solid-phase peptide synthesis
the broader needs and interests of the industry. This includes (SPPS). Many greener solvents should find utility in this space
large molecules, which are the primary focus of the Roundtable but will require significant efforts as a majority of these
greener biologics team.2,3 In addition, an area of emerging
importance is the concept of “medium molecules”, typically
Special Issue: Excellence in Industrial Organic Synthesis 2019
synthetically produced, larger than standard small molecules,
and including modalities such as peptides. These “medium Received: November 24, 2018
molecules” can be roughly defined as APIs with a molecular
© XXXX American Chemical Society A DOI: 10.1021/acs.joc.8b03001

J. Org. Chem. XXXX, XXX, XXX−XXX
The Journal of Organic Chemistry Perspective
Figure 1. Greener coupling reagents.
processes are operated by manufacturers using decades-old 2. SUSTAINABILITY IN PEPTIDE SYNTHESIS

practices where green chemistry has not been an area of focus. 2.1. Improving Classical Synthetic Methodologies.
Also contributing to the poor environmental profile is the 2.1.1. Coupling Agents. Although formation of amide bonds is
pervasive and extensive use of chromatography to produce one of the most prominent transformations encountered in the
peptide products with required quality attributes. In 2016, the pharmaceutical industry, it is still lacking general and robust
ACS GCI Pharmaceutical Roundtable identified development catalytic methods. Remarkable reports from Beller, Milstein,
of greener peptide processes as a critical unmet need, and a Gooßen, and Sheppard paved the way for future develop-
new Roundtable team formed to address this important area. ment,26−30 but the road is still long to reach mature and viable
The initial focus of this group is to highlight current best catalytic protocols for the application in Active Pharmaceutical
practices in peptide synthesis and encourage much needed Ingredients (API). From a toxicologic and regulatory stand-
innovations. In this perspective, we aim to summarize the point only, it is required to be at very reduced residual levels of
current challenges of peptide synthesis and purification in transition metals in the API, typically at or below 10 ppm,
terms of sustainability and highlight possible solutions. which further increases the technical challenges.
Therefore, most amide coupling protocols in peptide
1. CLASSICAL APPROACHES TO CHEMICAL PEPTIDE synthesis currently involve the use benzotriazole derivatives.
SYNTHESIS Key examples of that are uronium/aminium salts HATU,
HCTU and TBTU; phosphonium salts PyBOP, PyAOP and
1.1. Liquid-Phase Peptide Synthesis (LPPS). Early PyClock; and the combination of carbodiimides with HOBt,
syntheses of peptides were performed in solution requiring HOAt or 6-Cl-HOBt.31,32 These reagents provide high
careful manipulation of protecting groups and multiple coupling efficiency and low amino acid racemization and are
challenging workups and isolations.6 The use of this method- stable as solids with some of them even stable in solution.
ology can be problematic for the synthesis of longer and more However, the benzotriazole motif has been reported to exhibit
complex peptides but remains valid for shorter peptides and explosive properties, making scale-up and work at high
fragments.7 temperatures difficult.33 In addition, these reagents present
1.2. Solid-Phase Peptide Synthesis (SPPS). The seminal very poor atom efficiency.
work of Bruce Merrifield introduced solid-phase peptide Unfortunately, there is no general solution yet for the green
synthesis (SPPS), greatly simplifying the removal of non- formation of amide bonds during peptide synthesis, especially
tethered entities like reagents and solvents.8−10 This approach for long peptides. One of the big challenges is that peptide
opened the doors for automation as subsequent couplings complexity has increased significantly in the past decade with
could be iteratively performed by a defined set of unit many peptide products now containing >30 amino acids.34
operations.11−14 As a result, many challenging peptides have The field is too extensive to be covered in detail in this
been synthesized using SPPS.15−18 Originally, N-α-Boc- Perspective with new coupling reagents constantly developed
protected amino acid building blocks were used (Boc/Benzyl without their sustainability properties fully tested. Relevant
strategy). Subsequently, the Fmoc/tBu strategy gained popular- examples of reagents appearing to be more sustainable are
ity mainly because it avoided the use of hazardous HF for depicted in Figure 1. Ethyl cyano(hydroxyimino)acetate
cleavage from the solid support and final deprotection.19,20 “oxyma pure”,35 which is used in combination with a
However, SPPS is far from perfect, especially in terms of carbodiimide, and its derived uronium salt COMU36 generally
greenness. Typical syntheses require an excess of amino acids, show equal or better coupling efficiencies and lower amino
coupling reagents and base to achieve maximal conversion at acid racemization than their benzotriazole counterparts. One of
each step. In addition, Fmoc amino acids can be considered to the limitations of COMU is that its application to automated
have lackluster green credentials in that they have very poor solid-phase peptide synthesis is very limited due to its
atom economy. Often overlooked is the use of large excesses of instability in DMF. However, a recent study has shown that
piperidine (>20 equiv) to drive Fmoc removal to completion. COMU is relatively stable in γ-valerolactone, which makes it
These processes also rely on the use of large excess of solvents potentially more attractive for greener automated solid-phase
to wash the resin after each operation. This results in increased peptide synthesis.37 Finally, it has to be noted that another
process mass intensity (PMI: ratio of total mass of all input recent publication, reporting differential scanning calorimetry
material over mass of isolated product) and much higher cost (DSC) studies, classified COMU in the group of “least
of goods than for small molecules. Syntheses of hydrophobic preferred” reagents for peptide production.38 Hence, the
peptides pose additional challenges since aggregation of practicality of COMU is still in question.
growing peptide chains is a common issue.21,22 Often, Other, less used, but relatively green coupling agents are
specialized resins, solvents, and lower resin loading need to TFFH and its derivatives, which can generate amino acyl
be employed, thus further impacting the process greenness and fluorides in situ and have been applied in a number of solid-
cost of goods.23−25 phase peptide syntheses,39 and propylphosphonic anhydride
B DOI: 10.1021/acs.joc.8b03001
(T3P), which is stable in solution and can potentially be used highlighted was the higher viscosity of NBP versus established
in SPPS.40 solvents such as DMF, although heating was proposed as a
The current state of the art for commercial synthesis of large practical way to overcome this issue during synthesis
peptides still predominantly uses DIC/HOBt. However, the procedures. Nevertheless, despite the encouraging results of
use of “oxyma pure” has gained significant traction in industry. alternative solvents most exemplifications are for very short
Finally, alternative ways of forming amide bonds have also model peptides. The publication of synthesis of larger peptides
been proposed aiming to improve atom efficiency and to exemplify these findings should help industrial uptake.
compatibility with various solvents among other properties.41 Achieving SPPS in water would address major sustainability
Most of these methods are not as green as desired or cannot issues associated with toxic and hazardous solvent usage.
fully be applied to peptide synthesis yet, but we believe that Although there is no general solution yet, there are some
this type of research will pave the way to greener solutions. successful cases, both at room temperature.47−50 For instance,
Considerations will also need to be made on the impact of EDC-HONB as coupling reagent, aided by the presence of
these potential novel approaches to the starting material supply zwitterionic detergent and microwave irradiation to couple
chain, e.g., Fmoc-amino acids. certain Boc-protected amino acids, was used to successfully
2.1.2. Solvents. Solvents represent the vast majority of the synthesize Leu-Enkephalin peptide in water on a PEG-based
waste generated in chemical processes, and this is especially resin.48 Fmoc-amino acids in nanoparticulate form have also
relevant for solid-phase peptide synthesis. Hence, using greener been used to successfully carry out SPPS in water.51 Several
solvents or finding ways to reduce or recycle solvents are of water-soluble protecting groups to aid in aqueous SPPS have
high interest. The main solvents used for solid-phase peptide been described and recently sulfonated versions of Fmoc and
synthesis are DMF, NMP, and in lower amounts, dichloro- Boc protecting groups have been patented.49,52 Furthermore, a
methane, diethyl ether, and tert-butyl methyl ether. All these hybrid approach has been proposed to perform the coupling
solvents present various hazards. Most importantly, DMF and and deprotection steps in organic solvent and washes in
NMP have reproductive toxicity hazards and are likely to be aqueous solvent to improve the greenness of the process while
restricted or authorized by the European Regulation for minimizing side-reactions associated with aqueous synthesis.53
Registration, Evaluation, Authorisation and Restriction of Finally, we believe that safer and greener nonfluorinated
Chemicals (REACH), which may eventually preclude the use replacements for hazardous trifluoroacetic acid are also
these solvents.42 required.
Thus, there is a need for research on new green solvents 2.1.3. Resins Contributing to a Greener Solid-Phase
applicable to peptide synthesis as well as on new synthetic Peptide Synthesis. Resin recycling has been mainly described
strategies to make peptide chemistry compatible with green for the 2-chlorotrityl resin, but to our knowledge, that is still a
solvents. The ideal solvent should be able to dissolve all very small research field.54−56 An important characteristic of
peptide synthesis reagents, enable efficient amide bond many of the new greener resins (i.e., polyethylene glycol-
forming reactions and deprotections, and swell the solid- based) is that they swell to a significantly greater extent in most
support of choice. solvents and especially in trifluoroacetic acid. This makes the
In recent years, a number of greener solvents have been used solvent required per mass of resin much larger. The poly-ε-
for peptide synthesis. For instance, 2-MeTHF (2-methylte- lysine resin SpheriTide is less compatible with green solvents
trahydrofuran) has been used for the solid-phase synthesis of in comparison with polyethylene glycol-based resins, according
Aib-enkephalin pentapeptide on a polyethylene glycol based to recent reports. However, it has a high-loading and is
support (ChemMatrix). Coupling conditions were DIC/ biodegradable, and starting materials for the resin are derived
Oxyma Pure in 2-MeTHF, and the Fmoc group was removed from renewable resources.45 We believe that the ideal resin
with 20% piperidine in in 2-MeTHF.43 More recently, γ- should swell well enough for reactions to occur efficiently but
valerolactone has been used as a replacement for DMF for the not swell more than is required and ideally should be
SPPS of Aib-ACP decapetide on a ChemMatrix resin.37 In this compatible with greener solvents.
case, DMF was still used for the washings and in the Fmoc 2.1.4. Chemical Ligation. Since the development of the first
removal step. However, the same authors describe successful native chemical ligation (NCL), the merging of unprotected
Fmoc removal using 20% piperidine in γ-valerolactone on both peptide fragments has permitted the synthesis of long peptides
polystyrene and polyethylene glycol (ChemMatrix) resins.44 and proteins that were previously impossible or very
Interestingly, the authors also show that the most common challenging to synthesize.57,58 This convergent approach can
Fmoc-amino acid derivatives are soluble in γ-valerolactone at potentially afford higher yields, purer crudes, and reduced
>0.1 M, which makes the solvent of potential use for usage of solvents. In addition, most ligation reactions are
automated synthesis. performed in aqueous conditions.
Propylene carbonate has been used for peptide synthesis in More recent applications of native chemical ligation include
solution (Boc/Bn strategy) and for the manual solid-phase a synthesis of the enzyme Sortase A and of isotopically labeled
synthesis of nonapeptide bradykinin (Fmoc/tBu strategy) on human insulin.59−61 SPPS is primarily used to access both the
the ChemMatrix resin. Solid-phase coupling times were longer C-terminal thioester containing peptide as well as the N-
than usual (2 × 1 h), but in this case, both coupling and Fmoc terminal Cys-containing peptide fragments.62 As an extension
removal were performed in propylene carbonate.5 The same of NCL, expressed protein ligation (EPL) involves recombi-
group has published a thorough study of the swelling of various nant expression to generate either peptide or protein
resins in green solvents which we envisage will be extremely fragments. Thus, protein bearing C-terminal thioester can be
useful for future green syntheses of peptides.45 ligated with a synthetic peptide fragment with N-terminal Cys,
Recently, an in-depth study of DMF replacements for SPPS allowing access of semisynthetic non-native proteins with
has highlighted N-butylpyrrolidinone (NBP) as suitable useful applications.63,64 Overall, the progress in green ligation
alternative for certain applications.46 One of the drawbacks methodology in academia has been outstanding since the early
C DOI: 10.1021/acs.joc.8b03001
Figure 2. (a) Native chemical ligation; the reacting peptide/protein fragments can be recombinantly accessed in expressed protein ligation. (b)
Traceless Staudinger ligation. (c) Ser/Thr ligation (R = H/Me). (d) KAHA ligation (Opr version).
1990s. However, there has been very little traction in industry. • KAHA ligation: A peptide bearing an α-ketoacid at the
Key improvements that may encourage industrial uptake are C-terminus forms a native amide bond with another
the development of improved resins and synthesis of bearing a hydroxylamine at the N-terminus.73 A recent
thioesters. In addition, development of green methodologies example is the synthesis of the highly hydrophobic
to desulfurize cysteine to alanine during ligation could improve antiviral membrane-associated protein IFITM374
scope significantly.
Finally, several promising forms of cysteine-free peptide 2.1.5. Advances in Rapid Synthesis at a Small Scale.
chemical ligation have been featured in recent reviews (Figure Syntheses of peptides by confining reagents and solvents onto
2).62,65 specific areas of Teflon-patterned paper allowed for rapid flow-
through syntheses of peptide libraries.75 The solvent-repelling
• Staudinger traceless ligation: A peptide thioester bearing Teflon barrier on the paper localized various reagents and
a phosphine forms an amide bond by reaction with an washes in the uncoated part of the paper, enabling parallel
N-terminal azide.66,67 This ligation has recently been flow-through synthesis, which were also amenable to infrared
applied to the synthesis of glycan−peptide conjugates.68 radiation (IR) heating to improve coupling efficiency.76 This is
• Ser/Thr ligation: Peptides bearing a C-terminal o- an innovative example of sustainable synthesis of peptide
salicylaldehyde ester form amide bonds with peptides libraries for screening purposes. Fluorous mixture synthesis
containing N-terminal Ser or Thr via an oxazolidine (FMS) had been successfully applied to synthesize libraries of
intermediate.69,70 Recent examples are the syntheses of tri- and pentapeptides using split-type liquid-phase combina-
teixobactin and analogues.71,72 One point that needs to torial synthesis using fluorous-Fmoc (f-Fmoc) reagents as
be addressed is that the standard ligation protocols encoding tags to allow for their rapid separation.77,78 This is a
requires the use of pyridine/acetic acid (6:1). good example of divergent polypeptide synthesis, providing
D DOI: 10.1021/acs.joc.8b03001
reliable access to large numbers of peptides. Apart from Innovation Scorecard that is freely accessible on the ACS Web
advances in linear peptide generation, libraries of cyclic site.95 Moreover, the scorecard quantifies the innovation
peptides were also obtained by NCL methods.79 Finally, a impact of process scientists by the degree of RPG upgrade
convergent SNAr-based diversity-oriented side-chain macro- and amount of waste reduction of the current manufacturing
cyclization with Cys residues and perfluoroaryl linkers allowed process vs an earlier version. As a starting point, contract
rapid synthesis of macrocyclic peptide libraries.80 manufacturing organizations (CMOs) and pharmaceutical
2.1.6. Peptide Production. 2.1.6.1. Overview. With growing companies are encouraged to start collecting PMI/cEF data
interest of peptides in the pharmaceutical industry, kilogram- for their manufacturing processes.
scale syntheses have become relatively common.81 On a In order to appreciate the amount of cogenerated waste in
production scale, besides coupling efficiency, reagent choice is peptide versus small-molecule synthesis and meaningfully rate
also dictated by safety and price. The equivalents of reagents its RPG, one should consequently apply iGAL methodology.
are carefully optimized to minimize the cost associated with However, a barrier to analysis is iGAL’s $100/mol starting
routine use of excess inputs in SPPS. To reduce the hazardous material rule, since purchased peptide building blocks typically
footprint and lower costs during multikilogram scale exceed this “commodity-type” limit, and significant effort is
production of peptides, research is often dedicated to replacing required to determine the waste associated with the production
toxic reagents like carbodiimides with simpler and safer of those building blocks. Fortunately, with peptides, unlike
reagents. A nice example is the use of acyl chloride and with small molecules, we are dealing with a limited number of
imidazole to effect amidation for production of an HIV common building blocks. Thus, a feasible solution is to create
protease inhibitor candidate.82 Often, reagent equivalents are an iGAL inventory of intrinsic cEF of the common peptide
lowered and double couplings are eliminated while extending building blocks to standardize and facilitate analysis. The ACS
coupling times to achieve the same efficiency and reduced GCI PR is undertaking such an effort to make common amino
waste generation. It is common in the smaller academic scale acid iGAL inventories available on its Web site. The results of
and in early drug discovery space to employ anywhere between this iGAL analysis of peptide drugs will be reported in due
2 and 10 equiv of reagents to ensure near-complete couplings, course. Future efforts of the roundtable will also target
while manufacturing processes for peptides typically demand collecting metrics data for peptide drug manufacturing
use of no more than 30−50% excess reagents during each processes and reporting the data.
coupling. Often for smaller peptide targets (<10 amino acids), 2.2. Novel Peptide Synthesis Methodologies. Linear
solution-phase chemistry is optimized to minimize the reagent SPPS has a general loss in efficiency due to failure modes in
waste. However, isolation and purification of intermediates can synthesis, especially for sequences >15 amino acid residues.84
be challenging even for moderately long targets. There have This has a consequential impact on product purification
been reports of continuous solution-phase peptide synthesis complexity and overall process yield. Further to this,
which avoids the purification challenges of traditional solution hydrophobic amino acid sequence issues can put limits on
chemistry by leveraging buffered aqueous extractions of overall yields. This can often be attributed to an on-resin
reagents and water-soluble Fmoc-deblocking adducts.83 To aggregation phenomenon. These problems can be surmounted
enable higher volume production, hybrid SPPS/LPPS is often in the majority of cases by employing the combined peptide
used in production. For example, an HIV membrane fusion expertise, experience, and knowledge gathered over decades of
inhibitor, Enfuvirtide/T20, a 36-amino acid peptide was intense SPPS investigations. However, lengthy linear syntheses
produced commercially at a scale of several tons a year by a have a high intrinsic risk as a single mistake can result in a total
hybrid solution- and solid-phase chemistry.84 Other examples loss. Failure rates for the chemical synthesis of large peptides
include hybrid SPPS/LPPS processes for exenatide and (>30-mer) at contract manufacturing organizations is
liraglutide.85−87 Often, multiple routes and processes are estimated to be approximately 20%. The chemical ligation
developed to address the challenges of peptide synthesis on methodologies and hybrid SPPS/LPPS strategies mentioned
scale. A paradigmatic example of this is the synthesis of above can solve some of these problems but there is clearly still
octreotide, a cyclic octapeptide SRIF agonist.88 a need for alternative approaches and technologies that could
2.1.6.2. Metrics for Peptide Synthesis. The waste ultimately give overall efficiency gains and reduce the
cogeneration in peptide production has been reported to be environmental impact of peptide synthesis and produc-
in the multiton range for each kilogram of produced tion.7,96,97
peptide89,90 vs hundreds of kilograms for small-molecule 2.2.1. Peptide Synthesis in Flow. Flow chemistry has green
synthesis.91 credentials due to fast heat transfer leading to reduced energy
A proven management adage is that “you can’t manage what usage and reaction times, which reduce overall environmental
you don’t measure”. However, despite the clear need for impact. A recent renaissance in SPPS flow investigations for
metrics in peptide manufacture there has been no notable peptide synthesis98,99 has been apparent due to the swift
application thereof. Process Mass Intensity (PMI)92 and development and implementation of packed-bed flow SPPS for
complete E factor (cEF)93,94 have emerged as the leading small scale peptide and protein synthesis.100 The accomplish-
measures for coproduced process waste in small molecule drug ments, including ultrarapid synthesis of complex proteins101
synthesis, and were recently integrated as foundational and full system automation,102 have reinvigorated a field of
elements with the Innovation Green Aspiration Level investigation that started the following decade after Merrifield’s
(iGAL).91 iGAL represents the first standardized process SPPS concept took root in research laboratories103,104 and
waste analysis using a $100/mol starting material rule, it focused on online feedback, reduction of material usage, and
quantifies Relative Process Greenness (RPG = iGAL/cEF) by automation.105 Until now, there have been very few reports of
drug complexity- and development phase-adjusted comparison scalable SPPS in flow,106,107 although attempts are being made
of a process against industry averages, and as such, it can to rectify this using a continuous band approach, feeding
output a fair green process rating via a Green Chemistry through sequential specifically designed reactor units.108
E DOI: 10.1021/acs.joc.8b03001
LPPS in flow has also been investigated to enable the swift aqueous washes. This was achieved by using DBU for Fmoc
synthesis of small peptide fragments in a fast, efficient and low removal in the presence of thiomalic acid to form water-soluble
waste manner.109,110 More focused on scaling-up, the use of dibenzofulvene adduct.123
low-cost, improved atom efficiency amino acid activation via Finally, a related approach has been describedgroup
N-carboxyanhydrides (NCAs) was investigated. This demon- assisted purification (GAP) employing a triphenyl phosphine
strated high throughput, productivity, and very significant PMI oxide “Tag” to allow precipitation of growing peptide chains,
reduction (>100×) for di- and tripeptide synthesis using including the potential to recycle the “Tag” potentially
continuous stirred-tank reactor (CSTR) setup and aqueous reducing overall waste during synthesis.124
reaction media.111 2.2.3. Organic Nanofiltration/Membrane-Based Reactor
An attractive option is to potentially utilize flow chemistry Systems. The development of membranes with greater organic
for hybrid SPPS/LPPS peptide processes. Production of small solvent compatibility125 has reignited the investigation into
fragments on resin (<10-mers) would likely be ideal process membrane-enhanced peptide synthesis (MEPS) to remove
intermediates or regulatory starting materials in that these side products and reagents from peptide synthesis,126 which
materials would be of high purity, generally stable, and require had first been investigated in the 1970s.113 A PEG Tag was
no specialized shipping or storage conditions. These fragments again used for the Tag-assisted LPPS, and several different
would be cleaved from the resin and coupled to other membrane types were used in a cartridge system under
fragments by LPPS. The Enfuvirtide/T20 manufacture is an constant volume diafiltration in between coupling and Fmoc-
example of a batch process utilizing this general strategy.84 removal cycles. This was demonstrated on two small peptides
2.2.2. Greener Tag-Assisted LPPS (Tag LPPS). Shortly after (4- and 5-mers). Indications were seen in this work for
Merrifield’s seminal SPPS contribution to the peptide synthesis potential to reduce the high solvent waste burden produced
field, the limitations of the newly invented methodology during peptide manufacture. This solvent waste reduction has
(principally side reactions and large reagent excess to been further investigated and demonstrated by a separate
overcome solid phase-matrix diffusion and achieve desired group in the context of a high-dilution peptide cyclization
high coupling conversions) drove investigation into the use of reaction.127
modified LPPS approaches using soluble “Tags”, such as Recently, published work in this area focused on MEPS
polystyrene (PS, no cross-linking)112 and polyethylene glycol process parameter understanding to control and reduce failure
(PEG),113,114 to provide solubilizing anchors for growing modes in the synthesis.128 Also, further development of the
protected peptide chains leading to homogeneous solutions “Tag” used in MEPS has been published, going beyond linear
kinetics potentially reducing need for large excesses of reagents polymers (such as PEG, PS) to branched anchors, with the aim
used in SPPS. The removal of side products and reagents in of reducing permeance of the growing peptide chain and, thus,
between coupling cycles (achieved by resin washing in SPPS) reduce yield loss through the membranes.129
was achieved via precipitation in the case of soluble PS or 2.2.4. Enzymatic Peptide Ligation. Chemo-enzymatic
crystallization in the case of PEG. Also, the first proposed use peptide synthesis has been an area of investigation for many
of membrane technology via ultrafiltration (UF) was years, promising high specificity,130 mild reaction conditions,
demonstrated,113,114 although UF cycle times appear to have and reduced waste streams. Various ligases like sortase,
been too long to be tolerable due to solvent-membrane butelase, peptiligase, or omniligase enable chemoselective
incompatibility issues.115 peptide ligation. Recently, this has been refined and
The Tag-assisted LPPS concept has been further developed demonstrated by EnzyPep B.V. to catalyze efficient peptide
by various groups in the past decade.116−119 These groups took cyclization and traceless condensation of unprotected peptide
inspiration from the earlier work described above and from the fragments for the synthesis of medium to large peptide
prior use of defined-structure lipophilic “Tags” in combinato- targets.131,132 The tangible benefit of this fragment con-
rial chemistry.120 There remain questions surrounding the densation approach is the use of unprotected peptide
broadness of the scope of this approach for peptide synthesis, fragments in predominantly aqueous coupling conditions,
specifically, whether the selected “Tag” can provide the circumventing solubility issues that have hampered more
required universal solubility for peptide elongation. This conventional chemical fragment condensation approaches.
scope question, can be observed through two elastin peptide This has been elegantly demonstrated by synthesis of thymosin
synthesis articles. The first one, by Chiba et al., indicated that, α1 and exenatide.133
in practice, physical pressure was required (using connected 2.2.5. Mechanochemistry. Mechanochemistry promotes
glass syringes) to mobilize the growing elastin peptide chain reactions between solids by induced mechanical energy (e.g.,
enough to carry out the synthesis.121 An article in response was by grinding all reactants in a ball mill), and the area has been
then published by Kent et al., who argued that the universal expanding rapidly since 1990.134−137 The solvent-free synthesis
solubilizing power provided by the cross-linked solid support or minimal solvent synthesis (liquid-assisted grinding) was
would mean SPPS would remain the dominant paradigm for evaluated for short peptides (two to five amino acids) using
peptide synthesis.122 Nevertheless, it has also been reported urethane-protected α-amino acid N-carboxyanhydrides
that the “Tag LPPS” approach can achieve >12-fold reduction (NCAs), Boc-protected α-amino acids, or N-hydroxysuccini-
in solvent waste for a 20-mer peptide versus the equivalent mide esters by grinding with α-amino acids, amides, or esters
SPPS approach.123 This was achieved by increasing the in the ball mill.138−141 Moreover, reactive extrusion shows
lipophilicity of the anchor molecules, which were endowed evolution of mechanochemistry for short peptides (2−3 amino
with long alkyl chains as C-terminal protecting groups, acids) application using the principle of mechanochemistry
resulting in peptides synthesized where intermediates were and flow chemistry.142 These publications have demonstrated
isolated by solvent extraction instead of precipitation.123 In the that mechanochemistry is an environmentally benign alter-
same work, Fmoc removal conditions were optimized to native for the preparation of short peptides in high yields with
facilitate the removal of dibenzofulvene adduct by basic very low epimerization.
F DOI: 10.1021/acs.joc.8b03001
3. SUSTAINABILITY IN PEPTIDE ISOLATION AND most organic solvents. Disadvantages of this technique include
PURIFICATION heat generated from higher flow rates may decrease resolution
and not many studies involving peptides are available.
The purification and/or isolation of peptides is often the
3.3. Liquid−Liquid Extraction (LLE). LLE, also known as
bottleneck of the manufacturing process. In addition, large
solvent extraction, is the oldest and most widely used method
amounts of high-level aqueous/low organic waste are
to separate intermediates in solution-phase peptide syn-
generated. thesis.151 A series of modifications of LLE have been developed
3.1. High Performance Liquid Chromatography over the years in order to maximize extraction efficiency and
(HPLC). HPLC is the most common technique for purification reduce solvent consumption and production costs. Relevant
and counterion exchange of peptides.143 Reversed-phase (RP) examples are liquid−liquid extraction in continuous flow
and ion-exchange (IEX) chromatography are preferred for the system,152,153 microdroplet rotation in a hydrocyclone,154
purification of peptides giving high resolution, robustness, and stirred/agitated extraction column155 and mixer settler.156
reproducibility of output. However, the interactions of Nevertheless, these alternative LLE procedures have not been
peptides with column packings differ significantly from those extensively applied to the synthesis of peptides or the results
of small organic molecules. Currently on the market are mixed- have not been published.
mode chromatography (MMC) columns for peptide purifica- On the other hand, ELLE (enantioselective liquid−liquid
tion in which the solute has more than one interaction with the extraction) is commonly used for separation of enantiomers
stationary phase during the separation. These interactions are including amino acids.157 Also, an ionic liquid−organic solvent
not only hydrophobic or ionic but hydrophobic/ionic, extraction was applied to the purification of dipeptides and
hydrophobic/hydrophilic, hydrophilic/ionic (type I−IV tripeptides achieving excellent separation and taking advantage
mixed beads).144 Mixed-mode chromatography may be used of the recycling properties of ionic liquids.158
as an alternative or complementary technique to RP, IEX, or 3.4. Case Studies. 3.4.1. Etelcalcetide: Eliminating Ion-
normal-phase chromatography for purification of peptides.145 Exchange Chromatography by a Precipitation Process.
A similar technique to MMC is doped reversed-phase Peptides manufactured by SPPS are most often prepared as
chromatography (DRP). DRP chromatography uses different their TFA salt due to the nature of peptide cleavage and
ligands (one ion exchange ligand-type and one reverse phase reversed-phase purification techniques.159 Ion-exchange chro-
ligand-type) and has more advantages in comparison with matography is the standard approach to convert peptide TFA
MMC as the interaction mechanism between the solute and salts to pharmaceutically acceptable salts.160 In the case of
stationary phase can be altered by changing the doping Amgen’s etelcalcetide (Figure 3), the conversion of the peptide
concentration of one of the ligands. Second, it can be used in
attractive-repulsive mode, where the ion exchange groups are
in repulsive mode with regards to the analyte.146 Finally,
purification of the peptide was demonstrated using multi-
column counter-current solvent gradient purification
(MCSGP) chromatography. The internal recycling and
reseparation of the overlapping fractions increases yield and
productivity, while reducing solvent consumption.147
MMC and MCSGP techniques demonstrate faster separa-
tion, higher yields and a more productive process compared
with standard RP-HPLC. Additionally, they reduce the
generation of hazard waste, consumption of water, and reduce
the cycle time of the purification process. Figure 3. Structure of etelcalcetide.
3.2. Supercritical Fluid Chromatography (SFC). SFC
provides several advantages over traditional HPLC, such as TFA salt to the final HCl salt involves ion-exchange
speed, practical use of longer columns, a normal-phase chromatography using an acetate resin to form the acetate
retention mechanism, and a reduction in the use of organic salt, followed by several lyophilization cycles in dilute HCl to
solvents. SFC can be used for the purification of up to 40-mer remove acetic acid and exchange acetate with chloride. This
peptides, both linear and cyclic. It uses CO2 as a mobile phase two-step conversion process, while effective, is both energy-
with modifiers (e.g., methanol, ethanol, 2-propanol) and and volume-intensive, ion-exchange is inherently dilute, and
additives (e.g., TFA or ammonium acetate).148,149 Zheng et multiple lyophilizations are typically required in order to
al. claimed that the acidity of the mobile phase modifier was remove acetic acid. The entire two-step process can take weeks
critical and the amount of TFA additive varied with different to perform on commercial scale, and the necessary volumes of
polypeptides.149 SFC appears to be a promising technique in water and tremendous lyophilization capacity demand place a
relation to greener chromatography compared with HPLC, due major bottleneck on large-scale peptide manufacture, posing
to the possibility of recycling the CO2 used during SFC significant risk to meet commercial drug demand.
purifications (as yet, still an unresolved topic). The environ- Amgen and Bachem teamed up to develop a noteworthy
mentally friendly nature is displayed as SFC produces less than solution to the two-step, high-volume ion-exchange/lyophiliza-
one-third of the organic waste in comparison with current tion sequence in their commercial manufacture of etelcalce-
purification methods.150 This same work also reported a 5-fold tide.161 Characterization of both starting material and product
reduction in analysis time. Other advantages of SFC include revealed a significant solubility difference between the
the following: less toxic modifiers used, achieving better etelcalcetide−TFA salt and the desired HCl salt; the TFA
resolution with chiral compounds and special attributes of salt measured 100 mg/mL solubility in 2-propanol, compared
CO2, such as, nonflammable, nontoxic, and lower in cost than to <1 mg/mL solubility for the HCl salt in the same solvent.
G DOI: 10.1021/acs.joc.8b03001
By leveraging small molecule solubility and crystallization substantially pure peptide that has met the logic criteria during
engineering principles, the team was able to design a controlled the “catch−release” (CR) process. Peptides longer than 10
precipitation of the desired HCl salt in aqueous 2-propanol. amino acid residues (up to 20-mers were evaluated) exhibited
The etelcalcetide−TFA salt, as a solution in aqueous 2- purity of less than 20% by a traditional chloroacetate
propanol, was charged to a solution of HCl in 2-propanol;
displacement method, while CR afforded purities of 60−
etelcalcetide−HCl was then isolated via filtration. The end
result is a practical process that directly converts the TFA salt 91%. Overall, considering all tested peptides, purities up to
to the HCl final form. The new, greener isolation process 98% were observed via the CR process, thus reducing/
completely eliminates ion-exchange chromatography and 12 eliminating chromatography needs. This technology success-
lyophilization cycles per drug substance batch. By removing fully purged a full range of common peptide-related impurities
the need for ion-exchange, the water burden on the process such as hydrolysis fragments and truncated sequences. An
was significantly reduced; the original ion exchange process attractive feature is the ready amenability of “catch−release” to
used for clinical manufacture required over 3 L of water for automation for purifying multiple crude peptide samples using
every gram of drug substance. The process, on commercial a standard peptide synthesizer, thus greatly increasing
scale, was able to be performed in a standard reactor and filter
throughput of pure peptides. Additionally, amine-bridged
dryer. The direct salt conversion process led to a 10-fold
capacity increase for the manufacture of etelcalcetide. macrocyclic peptides were successfully purified by this
3.4.2. Chromatography-Free Purification of Cyclic Pep- technology.
tides. Macrocyclic peptides are of great importance in the
therapeutic landscape since their size and tertiary structure 4. CONCLUSIONS
renders them ideal in modulating protein−protein interac- In recent years, it has become critical to match the
tions.162 This brief case study will highlight an important
advancement to improve isolation of pure macrocyclic peptide sophistication of synthetic chemistry in the production of
through chemical innovation.163 By replacing solvent-intensive cutting-edge medicines with innovative approaches to reducing
chromatography, this technology hugely improves the green- their impact on the environment. As part of global efforts to
ness of accessing macrocyclic peptides. This is noteworthy limit the negative effects of chemicals on people and the
since chromatography- related solvent usage contributes to a environment, greater pressure is being applied to restrict or
significant portion of PMI. In this Bristol-Myers Squibb study replace commonly used reagents, solvents, and resins in
funded by a BMS innovation grant, full-length synthetic peptide research and manufacturing. In this regard, both
peptides are modified at the N-terminus with an orthogonal academic and industrial groups have produced relevant
dual-mode linker capable of first enabling a solid phase
advances in the field of greener peptide synthesis and
“capture” of linear peptide followed by a base-mediated
cyclization and simultaneous “release” of desired pure peptide purification. Nevertheless, green peptide chemistry is still a
macrocycle. Strain-assisted azide−alkyne cycloaddition relatively small research field and many of these advances take
(SPAAC) proved to be a general orthogonal capture a long time to be applied to peptide manufacturing. Hence, it is
technology that can be broadly applied to a diverse set of important to not only invest more resources in the field of
target peptides of various sizes (5−20 membered demon- green peptide chemistry but to establish more synergies
strated) (Figure 4). between academia and industry, as well as between the
This approach is thus a “logic-test” where species from the pharmaceutical industry and CROs/CMOs. Development and
crude peptide milieu not tethered to the linker are rinsed away reporting of appropriate peptide process environmental
during the “catch” and (akin to a logic gate) the macro-
metrics, which is presently a gap, will enable identification of
cyclization-induced “release” step removes peptidic impurities
lacking a suitable nucleophile. The output is a sample of additional areas of improvement. Finally, it is important for
decision-makers to reconcile thoughts that greener approaches
are not only better for the environment but also more efficient
in terms of timelines and cost.
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected].
ORCID
Albert Isidro-Llobet: 0000-0001-9108-5916
Notes
Figure 4. “Logic gate” approach of catch−release purification. The authors declare no competing financial interest.
H DOI: 10.1021/acs.joc.8b03001
Biographies Subha Mukherjee earned his Ph.D. in Chemistry from the University
of Illinois at Urbana−Champaign in 2015. He worked under the
supervision of Professor Wilfred A. van der Donk in the areas of
peptide synthesis, bio-orthogonal chemistry, and chemical and
enzymatic synthesis to investigate lanthipeptide and phosphonate
natural products. He is currently a process chemist at Bristol-Myers
Squibb Co., where he enjoys developing scalable processes to enable
synthesis of small-molecule and peptide assets.
Albert Isidro-Llobet obtained his Ph.D. in Organic Chemistry (2008)

at the Institute for Research in Biomedicine, Barcelona Science Park,
University of Barcelona, under the supervision of Professors Fernando
Albericio and Mercedes Alvarez. His Ph.D. 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 Michael Kopach earned a Ph.D. in Organic Chemistry from the
joined GSK, Stevenage, UK, in 2012 as a peptide synthesis expert University of Virginia in 1995 under the guidance of Prof. W. D.
where he is currently an Investigator and GSK Associate Fellow in the Harman. He then completed postdoctoral studies in natural product
Chemical Biology department. synthesis at Colorado State University with Prof. A. I. Meyers. Mike
began his industrial career in 1997 at Roche, where his primary
responsibilities included development and implementation of
commercial processes for nelfinavir, Xenical, and enfuvirtidethe
first synthetic peptides produced on tonnage scale. In 2001, Mike
joined Eli Lilly and Company and has led several small-molecule
phase 1 to phase 3 R&D projects. Throughout his industrial career,
Mike has led research teams applying green chemistry principles to
product research and development and has published several
collaborative articles in this field. For the past decade, Mike has
represented Eli Lilly and Company at the ACS Green Chemistry
Institute Pharmaceutical Roundtable including serving a two-year
term as cochair from 2011 to 2013 and another from 2017 to present.
After a 14 year career within Lilly Small Molecule R&D, Mike now
has responsibility for multiple synthetic peptide projects. In this area,
Martin Kenworthy studied chemistry at University of Manchester Mike led an internal infrastructure build to bring peptide synthesis
(UK) and obtained his Ph.D. in 2003 with Prof. Jonathan Clayden. capability in house.
After postdoctoral studies with Prof. Richard Taylor at York
University (UK), he joined AstraZeneca in 2006 (Macclesfield,
UK), where he works in Chemical Development, within Process
Technology and Development.
Katarzyna Wegner obtained her Ph.D. in Organic Chemistry (2007),

Faculty of Chemistry, at the University of Gdańsk under the
supervision of Professor Zbigniew Grzonka. In 2008, she joined
IPSEN, Ireland, where she is currently Principal Chemical Process
I DOI: 10.1021/acs.joc.8b03001
Development Chemist responsible for improvement and innovation Frank Roschangar is director of process research and is based in
of novel peptide and small-molecule process manufacturing. Ridgefield, CT. He leads multiple teams of process and analytical
researchers to effectively manufacture exploratory drugs. He also
oversees the global external chemistry activities with respect to
supplier and intellectual property strategies, manages departmental
costs and resources, and routinely engages in continuous improve-
ment initiatives. Frank obtained a Ph.D. from Rice University in 1996,
followed by a Postdoc at Scripps, and an Executive MBA from Yale in
2014, and has over 50 publications and patents. After 4 years at GSK,
he joined Boehringer Ingelheim in 2002. Besides leading the global
green chemistry program, he is active in the IQ Green Chemistry
working group, cochairs the ACS GCI Pharmaceutical Roundtable,
and lectures the IQ-ACS green chemistry short course.
■ ACKNOWLEDGMENTS
This manuscript was developed with the support of the
Fabrice Gallou received his Ph.D. from The Ohio State University American Chemical Society Green Chemistry Institute
(2001) in the field of natural products total synthesis. He then joined Pharmaceutical Roundtable (https://www.acs.org/content/
Chemical Development at Boehringer Ingelheim, USA. He sub- acs/en/greenchemistry/industry-business/pharmaceutical.
sequently moved in 2006 to the Chemical Development group at html) The ACS GCI is a not-for-profit organization whose
Novartis, Switzerland, where he now is responsible for global scientific mission is to catalyze and enable the implementation of green
activities, overseeing development and implementation of practical and sustainable chemistry throughout the global chemistry
and economical chemical processes for large scale production of APIs enterprise. The ACS GCI Pharmaceutical Roundtable is
and new modalities. composed of pharmaceutical and biotechnology companies
and was established to encourage innovation while catalyzing
the integration of green chemistry and green engineering in the
pharmaceutical industry. The activities of the Roundtable
reflect its members’ shared belief that the pursuit of green
chemistry and engineering is imperative for business and
environmental sustainability.
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Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/joc

Sustainability Challenges in Peptide Synthesis and Puriﬁcation:

ABSTRACT: In recent years, there has been a growing interest in

T he American Chemical Society (ACS), the largest

© XXXX American Chemical Society A DOI: 10.1021/acs.joc.8b03001

Figure 1. Greener coupling reagents.

processes are operated by manufacturers using decades-old 2. SUSTAINABILITY IN PEPTIDE SYNTHESIS

Albert Isidro-Llobet obtained his Ph.D. in Organic Chemistry (2008)

Katarzyna Wegner obtained her Ph.D. in Organic Chemistry (2007),

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