large-scale synthesis of peptides

Large-Scale Synthesis ofPeptides

Lars Andersson1

Lennart Blomberg1

Martin Flegel2

Ludek Lepsa2

Bo Nilsson1

Michael Verlander3

1 PolyPeptide Laboratories(Sweden) AB,

Malmo, Sweden2 PolyPeptide Laboratories

SpoL, Prague,Czech Republic

3 PolyPeptide Laboratories,Inc., Torrance, CA, 90503 USA

Abstract: Recent advances in the areas of formulation and delivery have rekindled the interest ofthe pharmaceutical community in peptides as drug candidates, which, in turn, has provided achallenge to the peptide industry to develop efficient methods for the manufacture of relativelycomplex peptides on scales of up to metric tons per year. This article focuses on chemical synthesisapproaches for peptides, and presents an overview of the methods available and in use currently,together with a discussion of scale-up strategies. Examples of the different methods are discussed,together with solutions to some specific problems encountered during scale-up development.Finally, an overview is presented of issues common to all manufacturing methods, i.e., methods usedfor the large-scale purification and isolation of final bulk products and regulatory considerations tobe addressed during scale-up of processes to commercial levels.© 2000 John Wiley & Sons, Inc.Biopoly 55: 227–250, 2000

Keywords: peptide synthesis; peptides as drug candidates; manufacturing; scale-up strategies

INTRODUCTION

For almost half a century, since du Vigneaud firstpresented his pioneering synthesis of oxytocin to theworld in 1953,1 the pharmaceutical community hasbeen excited about the potential of peptides as “Na-ture’s Pharmaceuticals.” Further discoveries, includ-ing Merrifield’s solid-phase synthesis (SPPS) meth-od,2 introduced a decade later, recombinant tech-niques for expressing peptides and proteins inmicroorganisms,3 and most recently methods for pro-ducing peptides and proteins in transgenic animals4

and plants,5 have all combined to increase the avail-ability and lower the cost of producing peptides. Formany years, however, the major obstacle to the suc-cess of peptides as pharmaceuticals was their lack oforal bioavailability and, therefore, relatively few pep-tides reached the marketplace as approved drugs. As aresult, several major pharmaceutical companies aban-doned their research efforts in the area, in favor ofsmall molecule mimics of peptide or protein leadcompounds. In recent years, though, advances in theareas of formulation and novel delivery systems haverevitalized the field, leading to several highly success-

Correspondence to: Michael Verlander.Biopolymers (Peptide Science), Vol. 55, 227–250 (2000)© 2000 John Wiley & Sons, Inc.

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ful “blockbuster” peptide drugs, such as the luteiniz-ing hormone releasing hormone (LH-RH) analogues,leuprolide (Lupron), and goserelin (Zoladex). Theresulting resurgence of interest in peptides as phar-maceutical products has provided once more a chal-lenge to the peptide industry to develop economicallyviable methods for manufacturing relatively complexpeptides in large quantities. The challenge is beingmet, and a number of new peptide drugs are now inlate-stage development with target requirements, atcommercial launch, of up to hundreds of kilograms oreven metric tons.

While, as noted above, a variety of methods arenow available for the commercial-scale manufactureof peptides, chemical synthesis is still the most uni-versal approach, since it permits access to all possiblesequences, including those that contain unnaturalD-amino acids or amino acids that are completely syn-thetic in origin, which is not routinely possible withmethods based on biotechnology. Furthermore, it isnoteworthy that two of the pioneers of the field—duVigneaud and Merrifield—were both awarded NobelPrizes for their work on chemical synthesis of pep-tides by solution-phase and solid-phase methods, re-spectively. The general technology of chemical syn-thesis approaches is the subject of a recent, compre-hensive review.6 This article, therefore, will focus onthe practical aspects of the application of chemicalsynthesis methods to the large-scale production ofpeptides. First, a brief overview of the approaches,which are available and in use currently for large-scale manufacturing, will be presented, together witha discussion of strategies for scale-up. Each of thecommonly-used strategies will next be discussed inmore depth, together with selected examples. Finally,an overview of the methods used for large-scale pu-rification and isolation of final, bulk products will bepresented, together with a discussion of regulatoryissues commonly encountered during scale-up of pro-cesses to commercial levels.

STRATEGIES

Solution-Phase Methods

It is not surprising that, with almost 50 years ofexperience since du Vigneaud first published the syn-thesis of oxytocin,1 most of the approved peptidepharmaceuticals in use today are manufactured usingsolution-phase methodology. While the method hasbeen shown to be useful for the synthesis of very longpeptides and even small proteins, as can be seen fromTable I, the longest peptides manufactured for com-

mercial use by this method are the calcitonins (32amino acids). More commonly, the method is used toproduce small- to medium-length peptides in quanti-ties of up to hundreds of kilograms or even metrictons per year. Such products include angiotensin con-verting enzyme (ACE) inhibitors, the dipeptidessweetener, Aspartame, HIV protease inhibitors, oxy-tocin, desmopressin, and LH-RH analogues, such asleuprolide and goserelin.

Solid-Phase Methods

When Merrifield first introduced the solid-phasemethod in 1963,2 his discovery was met with consid-erable skepticism in the scientific community. Thiswas based on comparison with the well-establishedsolution-phase methodology, in which intermediatesand fragments were routinely isolated, purified, andcharacterized. In contrast, in Merrifield’s solid-phasemethod, no purification was possible until after cleav-age of the fully assembled peptide from the solidsupport, when most of the by-products accumulatedduring the synthesis were simultaneously cleaved.Furthermore, when the solid-phase method was intro-duced, the analytical methods and purification tech-niques available to the peptide chemist were nowherenear as powerful or discriminating as those in routineuse today, and therefore the purity of the peptidesproduced by the Merrifield method was in question.Nevertheless, from the beginning, the techniqueproved to be an extremely valuable addition to theresearch chemist’s repertoire, especially since it couldbe automated, permitting rapid synthesis of relativelycomplex sequences. The subsequent introduction ofalternate protecting group strategies, particularly the9-fluorenylmethyloxycarbonyl Fmoc/t-butyl combi-nation,7 in place of the t-Boc/benzyl combinationoriginally proposed by Merrifield, increased the ver-satility of the method. Finally, as more powerful an-alytical methods and purification techniques becameavailable, especially those based on reverse-phasehigh performance liquid chromatography (RP-HPLC), the technique has been increasingly exploitedas a manufacturing method for a number of commer-cial products, such as LH-RH and analogues, soma-tostatin and salmon calcitonin (Table I).

Hybrid Approaches

While both the solution-phase and solid-phase ap-proaches have clearly proven to be effective for themanufacture of a relatively wide variety of products,each strategy has its limitations. For example, therelatively lengthy development times required for so-

228 Andersson et al.

lution-phase syntheses usually preclude the consider-ation of this technique for the manufacture of productsfor early-phase clinical studies, when the need forrapid production is essential, in order to confirm thefeasibility of proceeding further with the develop-ment of the product. On the other hand, whilesolid-phase methods are usually employed for themanufacture of supplies during the early stages of aproduct’s development, scale-up of the method iscommonly thought to be difficult or impossible,especially if the original Merrifield method, usingBoc strategy, is employed, because of the use ofstrongly acidic reagents, such as liquid hydrogenfluoride, to detach the peptide from the resin. How-

ever, with the introduction of new resins, such asthe 2-chlorotrityl chloride8 and SASRIN9 resins, itis now possible to synthesize protected peptidesand fragments that can be detached from the resinwith the protecting groups intact. This has openedup the interesting possibility of a “hybrid” ap-proach, in which the manufacture of complex se-quences is approached through the solid-phase syn-thesis of relatively large, protected fragments,which are subsequently assembled either by solu-tion-phase or solid-phase methods. While still notextensively utilized, this approach offers consider-able promise for the commercial-scale productionof large peptides, which would otherwise present

Table I Some Approved Peptide Pharmaceuticals and Their Methods of Manufacture

Peptide Length Methoda

Oxytocin 9 CVasopressin analogues

Pitressin 9 CLypressin 9 CDesmopressin 9 C, SPTerlipressin 12 C, SP

Atosiban 9 CAdrenocorticotropic hormone (ACTH) (1–24) 24 CInsulin 51 E, S, RGlucagon 29 E, SP, RSecretin 27 ECalcitonins

Human 32 CSalmon 32 C, SPEel 32 C, SPDicarba-Eel (Elcatonin) 31 C, SP

Luteinizing hormone–releasing hormone (LH-RH) and analogues 10 C, SPLeuprolide 9 CDeslorelin 9 SPTriptorelin 10 SPGoserelin 10 SPBuserelin 9 SP

Parathyroid hormone (PTH) (1–34) 34 SPCorticotropin releasing factors

Human 41 SPOvine 41 SP

Growth hormone releasing factor (1–29) 29 SPSomatostatin and analogues 14 C, SP

Lanreotide 8 SPOctreotide 8 C

Thyrotropin releasing hormone (TRH) 3 CThymosina-1 28 SPThymopentin (TP-5) 5 CCyclosporin 11 EIntegrilin 7 C

a Manufacturing methods: C5 classical (solution-phase) chemical synthesis; E5 extraction from natural sources; R5 recombinant; S5 semisynthesis; SP5 solid-phase chemical synthesis.

Large-Scale Synthesis of Peptides 229

significant challenges when manufactured by eitherconventional solution-phase or solid-phase meth-ods.

Scale-Up Development Strategy

Because of the need for rapid production, usually ofrelatively small quantities for toxicological and earlyphase clinical studies, expediency often dictates thatproducts be manufactured by solid-phase methodsduring early stages of development. Once the productprogresses past Phase 2, however, when the dose inhumans is defined, a scale-up strategy must be inplace. If the requirements for the product exceed thecapabilities of the solid-phase approach and a changein the manufacturing method is required, it is imper-ative that the strategy includes both consideration ofthe requirements imposed by the regulatory agenciesconsideration, as well as the technical issues associ-ated with scale-up. Therefore, the strategy must ac-commodate not only the projected requirements forthe bulk drug substance at product launch and beyond,but also the regulatory requirement to demonstratethat the product manufactured by the two methods ischemically and biologically equivalent, and meets thesafety profile established in toxicological and clinicalstudies. If these criteria are not met, the consequencescan be extremely costly, both in terms of delays inlaunching the drug, as well as the cost of repeatingtoxicology studies and even clinical trials. Since thedemonstration of chemical equivalence is most oftenbased on comparison of impurity profiles in the bulkdrug substance produced by the different methods, wepropose that, as far as possible, the two manufacturingmethods should be designed to use similar, or ifpossible, identical side-chain protecting groups on theindividual amino acids in the sequence. In this way,the final, protected precursor of the product will bevery similar, or preferably even identical, from bothmethods, and the same methods can be used for thefinal steps in the process, i.e. deprotection, purifica-tion, and final isolation. Using this approach, we be-lieve that it is possible to predict, with a high degreeof assurance, that expectations for demonstration ofequivalence can be met.

OVERVIEW OF THE SOLUTION-PHASEAPPROACH

Background

In the almost 50 years since the solution-phase ap-proach was first demonstrated,1 numerous advances

have been made, especially in the areas of protectinggroups and coupling methods, which increase theoptions available to the peptide chemist for the designof a solution-phase synthesis.6 When such a large-scale synthesis is first contemplated, therefore, con-sideration must first be given to thestrategyof theapproach, i.e., the general chemistry, especially thechoice of linear or convergent synthesis, the selectionof segments and their assembly, the (orthogonal) pro-tecting groups used, etc. As the method is developed,the specifictactics of the approach must be consid-ered, i.e., the problems that are of critical importancewhen manufacturing on an industrial scale, includingchemical, technical, economic, and safety aspects ofthe process, as well as the demands imposed by reg-ulatory agencies, all of which must be considered inpharmaceutical production. While all of these aspectsmay be more or less neglected on a laboratory (de-velopment) scale, any one may become limiting as theprocess is scaled-up to an industrial level.

Strategy

Linear vs Convergent Synthesis.The size of the pep-tide usually determines the strategy, and peptides upto five amino acids in length are generally manufac-tured by a linear strategy (i.e., stepwise addition ofeach amino acid in the sequence until the entire se-quence has been assembled), while for longer pep-tides a convergent strategy (i.e., synthesis of smallsegments or fragments that are subsequently assem-bled to give the final sequence) is more suitable. Thisis mainly due to “the arithmetic demon,”10 by which

FIGURE 1 Effect of yield per step on overall yield of asynthesis (the “arithmetic demon”6).


the overall yield in a multistep process is criticallydependent on the yield per step (see discussion underthe chemical and technical aspects section below andFigure 1). In addition, the choice of solvent is morelimited for longer peptides, which, in turn, limits themethods available for the workup of reaction mix-tures.

If a convergent strategy is utilized, the selection offragments is crucial, and as a guide, the followingfactors should be taken into consideration:

● Optimally, fragments should contain C-terminalGly or Pro residues, since this minimizes the riskfor racemization.11

● If it is not possible to select fragments containingC-terminal Gly or Pro, the C-terminal residueshould be an amino acid that is less prone toracemize, such as Ala or Arg.11

● Fragments should preferably be no longer thanfive amino acids.

Before embarking on a large-scale synthesis, it isclearly prudent to test several possible strategies on asmaller scale. With the increased availability of resinsthat can be cleaved under mild conditions, with side-chain protecting groups intact, a very convenient wayto accomplish this is to synthesize segments on a0.5–1.0 g scale, using solid-phase technology, andsubsequently couple them in solution. Besides givinginformation about the extent of racemization, thisprovides a hint about the solubility of the segments.Since the solid-phase method is very expedient, syn-thesis of the corresponding isomer, containing theC-terminalD-amino acid, is also recommended. Usingthis diastereomer of the desired peptide, analysis byHPLC of the extent of racemizaton during the cou-pling of the fragments is very facile.

Protecting Groups

a-Amino Group Protection.For large-scale solutionsynthesis, the most frequently used protecting groupsare the Boc and benzyloxycarbonyl (Z) groups, be-cause of the volatile by-products formed during thedeprotection step.6,11 Use of the Fmoc group is lessattractive, because of the lack of volatility and thereactivity of the dibenzofulvene by-product. The Bocgroup is removed by acids, and for large-scale pro-duction, the most common reagents are neat trifluoro-acetic acid (TFA) and HCl/ethyl acetate mixtures. TheHCl salts are often solids rather than oils, which is anadvantage if they are to be isolated. The Z group isremoved by reduction, usually by catalytic hydro-genolysiswith H2/catalyst, but also by reduction with

sodium in liquid ammonia. In the past, HBr in aceticacid was also frequently used for this purpose. How-ever, on a large scale, the fact that the hydrobromidesalts, which were isolated as precipitates from drydiethyl ether, were hygroscopic was often a disadvan-tage of this method.

Carboxyl Group Protection.The most suitable car-boxyl “protecting group” for large-scale solution syn-thesis is the free acid itself. This, of course, limits thechoice of coupling agents (see below). Many peptidesoccur as C-terminal amides, however, making protec-tion unnecessary. If protection is required, either thebenzyl or t-butyl esters are preferred. These are re-moved in the same manner as the Z group and Bocgroup respectively (see discussion above), so theymust be orthogonal to thea-amino protecting group.If an aliphatic ester (e.g., methyl or ethyl) is used,saponification, using NaOH or KOH, is the method ofchoice for deprotection, although this poses a risk forracemization, or in the case of hindered amino acidssuch as proline, the reaction rate could be very slow(sometimes even zero). However, an advantage ofthese simple esters is that they can be transformed intohydrazides simply by treatment with hydrazine, whichcan then be used directly for coupling via the azidemethod (see below).

Side-Chain Protection.In general, it is desirable touse the minimal side-chain protection approach forlarge-scale synthesis, since this will minimize thenumber of steps. However it is not possible to dis-pense with protection completely:

● The d- and e-amino functions of ornithine andlysine, respectively, must, of course, always beprotected, and as for thea-amino function, the Zand Boc groups are recommended.6,11,12

● The carboxyl functions of aspartic and glutamicacids must also be protected, preferably by thesame esters as those mentioned above fora-car-boxyl protection, i.e., benzyl ort-butyl es-ters,6,11,12 taking orthogonality into accountwhen choosing the side-chain protecting groups.

● The thiol function on cysteine may be protectedby the Acm group or as a benzyl sulfide, theformer group being removed by iodine with con-comitant disulfide formation, and the latter pref-erably being removed by reduction with sodiumin liquid ammonia.6,12 (Note: trityl is anotherpossible protecting group, and the combinationof Acm and trityl groups can be useful to facil-itate site-directed cyclizations.)


A disadvantage of the minimal side-chain protec-tion approach is that the peptide is necessarily morehydrophilic, which often complicates extractiveworkup procedures. In this case, global protectioncould be an alternate approach, provided that theprotecting groups have been chosen in such a way thatthey can be removed simultaneously, or at most, intwo steps.

Coupling Techniques

Coupling of Individual Amino Acids. Today, thepeptide chemist has a veritable arsenal of couplingmethods at his disposal, the most commonly used ofwhich are mixed carboxylic–carbonic anhydrides,13

carbodiimides (e.g., dicyclohexylcarbodiimide(DCC),water-soluble carbodiimide (WSCDI)),14 and uro-nium reagents (e.g., o-(benzotriazol-1-yl)-N,N,N9,N9-tetramethyluronium tetrafluoroborate (TBTU), o-(ben-zotriazol-1-yl)-N,N,N9,N9-tetramethyluronium hexaflu-orophosphate (HBTU)).15,16 In addition to thesemethods, commercially available preactivated aminoacid derivatives, such as active esters (e.g., hydroxy-succinimide (HOSu) and p-nitrophenol (HONp) es-ters),17 and N-carboxyanhydrides (NCAs),18,19 maybe used. These derivatives have the added benefits ofnot generating any by-products from the activatingagent and also of being compatible with an unpro-tected C-terminal amino acid residue in the aminocomponent. The latter is also the case for the azidecoupling method.20 For the carbodiimide and TBTUmethods, however, C-terminal protection is mandatory.

Coupling of Segments.When coupling segments, therisk for racemizaton is increased, due to the possibil-ity of oxazolone formation. However, several meth-ods are available for this type of coupling. One of themost common is the carbodiimide method (e.g., DCCor WSCDI ) with an added auxiliary nucleophile (e.g.,1-hydroxy-benzotriazole (HOBt), 1-hydroxy-azaben-zotriazole (HOAt), HOSu).21,22 Another reagent thathas become popular in recent years, due to increasedcommercial availability, is TBTU.15 The mixed anhy-dride method, using isobutyl chloroformate, with orwithout an added auxiliary nucleophile, is also usedfrequently, as is the azide method, due to the lowracemizaton associated with it. However, the N3

2 an-ion, produced as a by-product with the latter method,gives rise to safety concerns.

Tactics—Scale-Up and TechnicalConsiderationsThe discussion of strategy (above), which focused onthe chemical aspects of peptide synthesis, is, of

course, of a general nature and applies to both small-scale syntheses as well as to large-scale production.However, there are many technical factors that mustbe considered when a laboratory-scale synthesis istransferred into the manufacturing plant. These as-pects—the “tactics” of scale-up—are discussed be-low.

The term “large-scale synthesis” is, obviously,quite subjective. When performed in a research labo-ratory, a one-kilogram batch can be considered to bevery large scale, while the same quantity in a phar-maceutical plant usually is not. For the purpose of thisarticle, large-scale is defined as a range of kilogramsto metric tons. While the chemistry of any large-scalemanufacturing process does not differ from that usedon a small scale, a number of comments must be madeabout practical matters. Mostly these concern a com-bination of chemistry and technique. The final goal isto develop as efficient, cheap, and fast a procedure aspossible, but also equally important is fulfilling therequirements of the regulatory authorities. Althoughmany of these factors seem obvious, they are seldomdiscussed explicitly. Therefore, a short discussion ofsome of the more important factors is given below,with special emphasis on peptide synthesis.

Chemical and Technical Aspects.In any multistepprocess, it is clear that the number of steps should bekept at a minimum, in order to increase the total yield,as required by “the arithmetic demon.”10 Obviously, afifteen-step synthesis will be more expensive and la-borious than a ten-step synthesis, if the yields are inthe same range. However, if alternative syntheticroutes are available, a longer reaction sequence withhigher yields may be advantageous. Thus, a fifteen-step synthesis with a 90% average yield can give ahigher overall yield than a five-step synthesis with a70% average yield, or an equal yield to a seven-stepsynthesis with an 80% average yield (see Figure 1).On the other hand, much more work and time ispresumably required for a longer synthesis and, there-fore, careful evaluation of the overall economics ofthe process is required.

Closely related to the previous discussion is thequestion of whether to use a convergent or a linearstrategy. Typically, the synthesis of all longer indus-trial peptides is performed by segment condensations,i.e., by the convergent strategy. The reason for thischoice is, of course, the same as in the previousdiscussion. It should also be noted that using a con-vergent synthesis is advantageous from a “safety”aspect. In all chemical production, there is always apossibility of mistakes or failures, which may meanthat the batch has to be discarded. Obviously, in such


a case, it is better to resynthesize a shorter segment ina convergent synthesis than a longer sequence in alinear synthesis. As mentioned in the section on strat-egy, peptides of up to five amino acids in length areusually conveniently assembled and isolated by con-ventional methods, using extractions and precipita-tions. However, from a practical point of view it maybe advantageous to use even smaller segments of twoor three amino acids in length. The reason for this isthat it is often easier to obtain purer segments byprecipitation or even crystallization, which can beused to advantage in segment condensations. Second,use of smaller segments may shorten the productiontime because of the possibility of using parallel pro-duction equipment.

It should also be noted that small segments oftencan be assembled in two ways: either the normal, C3N direction, with both N- and C-terminal protection,or the other possibility, i.e., the N3 C direction, asillustrated schematically in Figure 2 for the tripeptidesegment, Boc–Phe–Pro–Gly–OH. As shown in thishypothetical example, it is possible, by assembling thepeptide in the N3 C direction and avoiding C-terminal protection, to shorten the synthesis by twosteps, which can be very important on a large scale.However, the risk of racemization and the conditionsfor performing the salt couplings in a practical mannermust be evaluated critically.

The use of extreme reaction conditions should beavoided in large-scale manufacturing processes.While it is easy and quite common to perform areaction in the research laboratory at230°C or even270°C, such conditions may cause problems on ascale-up, because the necessary equipment is expen-sive and also because longer cooling and heatingtimes are necessary, which restricts access to theequipment for other purposes. Fortunately, such con-ditions are seldom encountered in peptide synthesis,where reaction temperatures generally range between

220°C and1100°C. Other extreme reaction condi-tions, such as high pressure, very dry conditions, verylong reaction times, highly exothermic reactions, orthe use of very specialized equipment, are also seldomnecessary in peptide chemistry. However, it may benecessary to use toxic or hazardous chemicals such asTFA or hydrogen, which require special precautions.The high concentrations that are often required forsegment condensations can also give rise to stirringproblems, while the low solubility of fragments mayrestrict the scale of production because of limitationson reactor volumes.

As noted above, low solubility can create prob-lems, especially as the peptide chain increases inlength. For shorter peptides, ethyl acetate is the sol-vents of choice, due to its low toxicity, low boilingpoint, and lack of miscibility with water, making itwell-suited for extractive workup procedures. Forlonger peptides, DMF is recommended, which re-quires the use of alternate workup methods, such asprecipitation, for example with ethanol, rather thanextractive procedures.N-methylpyrrolidinone (NMP)and dimethylsulfoxide (DMSO) also have potentialfor use with longer peptides, but they remain to beexplored as solvents for large-scale solution synthesis.

An important consideration during scale-up is thetechnical feasibility of large-scale reaction and isola-tion procedures. This is a heterogeneous group of unitoperations and other procedures that can be usedconveniently on a laboratory scale but occasionallywill create serious problems in large-scale syntheses.Examples of such apparently trivial problems are asfollows:

● use of extreme reaction conditions (see discus-sion above);

● use of steel reaction vessels on a large scale(instead of the glassware typically used on alaboratory scale) that makes it difficult to see, forexample, color changes or separations in two-phase systems;

● use of solids, such as drying agents (e.g.,MgSO4), which requires unnecessary filtrationsand handling of solvents;

● precipitates or concentrated products that cannotbe removed as easily from a reaction vessel asfrom a glass flask;

● concentration under reduced pressure of aqueoussolutions from chromatographic purifications,which may be very time-consuming (an exampleof a problem that is more easily handled on alarge scale by the use of reverse osmosis, atechnique that is not as easily applied on a smallscale); and

FIGURE 2 Comparison of strategies for assembling thetripeptide, Boc–Phe–Pro–Gly–OH.


● because most unit operations on a large scale aremuch more time demanding than the same op-eration on a laboratory scale (for example, con-centration on a rotary evaporator at elevatedtemperatures can be carried out in minutes on alaboratory scale, while the same procedure on alarge scale requires many hours), side reactionsnot observed previously on a small scale mayoccur to a significant extent during such pro-longed operations.

A specific problem that is encountered on scale-upis the preferred requirement for isolated intermediatesduring the synthesis to be in the form of solids, ratherthan oils, which are difficult to handle and makein-process control complicated. This can be achievedin two different ways: by precipitation methods or bychromatography. Precipitation, or possibly crystalli-zation for small segments, is the preferred method,because it is generally quick and simple to performand easy to scale-up linearly, although it can some-times require a painstaking amount of work to findsuitable methods. On the other hand, chromatography,as a general technique, is demanding of time andresources on a multikilogram scale, with the currentlyavailable technology, at least, and is not possible toscale up in a linear manner. Nevertheless, a consid-erable degree of purification is usually obtained,which is sometimes difficult to achieve by other meth-ods, especially for longer segments and “difficult cou-plings,” which makes it a viable alternative to con-sider in such cases. For the final product, alternativepurification methods to chromatography seldom existat present.

Commercial and Economic Aspects.A considerablenumber of different protected and activated aminoacid derivatives and coupling agents are now avail-able commercially. However, due to the high cost andlimited availability of most of these in large quanti-ties, their use in industrial syntheses is often prohib-ited. For the same reasons, the use of minimum pro-tection strategies is also preferred. For example, theuse of side-chain protected arginine derivatives, suchas the costly 4-methoxy-2,3,6-trimethylbenzenesulfo-nyl (Mtr) or 2,2,4,6,7-pentamethyldihydrobenzofu-ran-5-sufonyl (Pbf) derivatives, may be preferredfrom a chemical point of view, but because of theirhigh cost, the inexpensive HCl salt is the preferredalternative. Similar considerations can affect thechoice of coupling additives, such as HOBt, 3-hy-droxy-3,4-dihydro-1,2,3-benzotriazine-4-one(HOOBt), and HOAt. Even if the latter is currently thepreferred reagent, especially for demanding cou-

plings,23 its cost is prohibitive on a large scale, andtherefore the least expensive reagent (HOBt) is nor-mally used. The choice of whether to use preactivatedor nonactivated amino acids is not so straightforward,however. While preactivated amino acids are moreexpensive, in situ activation procedures may be moretime-consuming and may give significantly loweryields and purities, which can offset the higher cost ofthe preactivated derivatives. Therefore, the choice be-tween the two types of starting materials can only bemade after the necessary development work has beencompleted.

It is not uncommon in published literature proceduresto use large excesses—for example, two or more equiv-alents—of the activated amino acid to complete reac-tions and increase the purity of the products. This prac-tice is unacceptable, obviously, from an economic stand-point on a large scale, and therefore reagents andreactants should be used in as close to stoichiometricamounts as possible, in order to minimize raw materialcosts, even if the purity may be adversely affected as aresult. Nevertheless, quality demands for final productsare equally important and the key challenge for manu-facturers today is to deliver a product both economicaland of consistently high quality.

Safety and Environmental Aspects.The reagents,chemicals, and solvents used in industrial processesmust not only be cheap and commercially availablebut also safe to use for both humans and the environ-ment. These considerations can often cause difficul-ties and add considerable costs to projects at thedevelopment stage because of problems such as thefollowing:

● elimination of diethyl ether, which is often usedas a convenient precipitation agent on a labora-tory scale, in large-scale processes, due to thehigh risk of explosions and/or fires;

● the need to find a substitute for dichloromethane,which is an ozone-destroying solvent;

● substitution of benzotriazol-1-yl-oxy-tris(dimeth-ylamino)phosphonium hexafluorophosphate (BOP)as coupling agent, because of formation of theknown carcinogen, hexamethylphosphoramide(HMPA), as a by-product;

● substitution of TFA and hydrogen fluoride (HF),both of which are highly hazardous;

● safe use of hydrogenation reactors for deprotec-tion reactions;

● elimination of HN3, a highly toxic by-product ofazide couplings; and

● control of NH3 emissions because of environ-mental pollution concerns.


In addition, the potential for runaway reactionsmust be minimized, although, fortunately, these areseldom encountered in peptide chemistry. In general,because of these concerns, when a choice of reagents,solvents, or reaction conditions exists, the less dan-gerous alternative must be utilized, even at the ex-pense of the yield or the purity of the product.

The minimization of hazardous waste, because ofenvironmental concerns and high disposal costs, mustalso be taken in account. Although it is usually notconsidered in these terms, the manufacture of pep-tides, as well as many other drugs, is, in reality, theproduction of large amounts of waste with the drug asa small side product, and the manufacture of onekilogram of peptide can easily result in 1000 L ormore of waste products.24

Examples of Large-Scale Solution-Phase Peptide Synthesis

Two processes for industrial-scale production of pep-tides will be discussed. The first of these relates to the

multikilogram scale manufacture of Atosiban drugsubstance, an oxytocin antagonist developed as a ther-apeutic product for treatment of preterm labor anddelivery.25 The other process relates to the multikilo-gram scale manufacture of Desmopressin drug sub-stance (l-deamino-8-D-arginine-vasopressin), an im-portant antidiuretic for treatment of diuresis, such asthat associated with diabetes insipidus, nocturnal en-uresis, and urinary incontinence.26

The Atosiban Process.The structure of Atosibandrug substance is shown in Figure 3 and the synthesisstrategy developed for the large-scale production pro-cess for manufacturing the product is outlined inScheme 1. It should be noted that, during the earlystages of development, the drug substance for the firstclinical trials was manufactured by a method based onSPPS. Subsequently, in order to meet the increaseddemands for drug substance, a change in manufactur-ing method from SPPS to solution-phase was consid-ered necessary. However, such a production changerequires that the impurity profiles of the old and newmethods are at least equivalent. In order to meet thisrequirement, it was decided to develop the solution-phase synthesis in such a manner that it shared acommon intermediate with the SPPS procedure, pref-

FIGURE 3 Structure of Atosiban drug substance.

SCHEME 1 Overview of the solution-phase synthesis of Atosiban drug substance.


erably the key intermediate of the SPPS process.Scheme 2 illustrates this approach, which has beenapplied successfully to the development of the pro-duction method for Atosiban, in which the commonintermediate of the two processes is the protectednonapeptide. The reaction steps following productionof the nonapeptide are common for both processes. Inthis context, it should be noted that the critical impu-rities of the two processes are formed in the subse-quent chemical steps, i.e., in the reduction and disul-fide bond formation, yielding an identical impurityprofile for both methods. As noted above, this strat-egy, utilizing a common intermediate, together withthe benefits of both SPPS and solution-phase synthe-sis, should be attractive to apply to the manufacture ofother drug substances, when requirements dictate theneed to change production methods from SPPS tosolution-phase.

Synthesis Strategy for Atosiban.The manufacturingprocess utilizesNa-Boc protected amino acids and aglobal protection strategy, with only a single aminoacid containing an unprotected side chain, i.e., that ofasparagine.27 The peptide chain is assembled by con-vergent synthesis, requiring, as indicated in Scheme 1,the synthesis of three separate segments—two dipep-

tides (I and III) and one pentapeptide (II)—which aresubsequently coupled sequentially to yield the pro-tected key nonapeptide intermediate (V). The dipep-tides are synthesized using the mixed anhydride cou-pling procedure,13 in which, prior to coupling, theamino acid derivatives are preactivated by treatmentwith isobutyl chloroformate. Fragment II is synthe-sized by stepwise coupling of preactivated Boc-aminoacid derivatives. Starting with unprotected proline,successiveN-hydroxysuccinimide ester couplings areperformed to yield the tetrapeptide. The pentapeptideis obtained by coupling Boc–Ile–NCA to the Bocdeprotected tetrapeptide rapidly (approximately 2 hreaction time) and in high yield. In this context, itshould be noted that we have successfully applied BocprotectedN-carboxyanhydrides, which are now com-mercially available, to the large-scale manufacture ofpeptides other than Atosiban. In spite of their rela-tively high cost, such activated amino acid derivativeshave proven to be highly efficient, since they reactcleanly and rapidly with nucleophiles such as thea-amino group of amino acids and peptides.17,18

Segment Condensations.The three segments (I–III)are assembled in such a manner that the risk of race-mization is minimized. To this end, the dipeptideMpa(Bzl)–D–Tyr(Et)–N3 (I) (Mpa 5 mercaptopropi-

SCHEME 2 Illustration of the development approach for Atosiban drug substance, utilizing acommon intermediate for solution- and solid-phase syntheses.


onic acid) is coupled to the pentapeptide (II) throughuse of an azide condensation, conditions known toyield low racemization in segment condensations.11

Likewise, the point of attachment of the heptapeptide(IV), produced in the previous segment coupling step,is a C-terminal proline, a residue less prone to race-mize in the DCC-HOBt mediated segment couplingutilized to yield the desired, protected nonapeptideintermediate (V).11

Deprotection and Cyclization. The protectinggroups of the nonapeptide are removed by reductionusing sodium in liquid ammonia28. This deprotectionmethod for benzyl protecting groups was applied bydu Vigneaud in his pioneering work on the synthesisof oxytocin1. It should be noted that it is possible toproduce kilogram batches of reduced peptide rou-tinely by this deprotection procedure, provided thatthe progress of the reduction is monitored carefully.The reduced peptide is oxidized to give crude Atosi-ban by treatment with iodine at a relatively highpeptide concentration (10 – 20 g peptide/liter) inaqueous acetic acid. Under these conditions, the oxi-dation reaction process takes place instantaneously,affording a high yield of cyclic peptide without sig-nificant formation of polymers.

Purification and Isolation. The crude Atosiban pep-tide is purified in a two-step chromatographic proce-dure employing ion exchange chromatography (IEC)and reverse-phase chromatography (RPC), which isdepicted schematically in Scheme 3. In the IEC stepon SP-Sepharose FF, the HPLC purity of Atosiban isincreased from about 90% to about 96%. Furtherpurification by RPC chromatography on C-18 derivat-ized silica, using a gradient of ethanol in acetic acid,

enhances the purity to 98% or higher. The purificationdata obtained by this method is summarized in TableII. It should be noted that the overall purification yieldof Atosiban drug substance after both chromato-graphic steps is greater than 80% and that no repro-cessing of side fractions is necessary during the puri-fication process. After the RPC step, the pool contain-ing pure peptide is concentrated by reverse osmosis(RO) and subjected to diafiltration, to remove ethanol,prior to the lyophilization step, which affords theacetate salt of Atosiban. The product has been char-acterized extensively and the impurity profile estab-lished, with identification of all impurities present indrug substance batches at levels greater than 0.1%(HPLC area %). Currently, this industrial-scale man-ufacturing method, which requires no chromato-graphic purification of peptide intermediates, is usedto produce approximately 20 kg of drug substance peryear, with future annual production estimated to be50–100 kg.

The Desmopressin Process.The large-scale solu-tion-phase process developed for Desmopressin drugsubstance (Scheme 4) resembles that described abovefor Atosiban drug substance. The structure of the drugsubstance is shown in Figure 4. Both compounds arecyclic nonapeptides containing a disulfide bond,formed during the cyclization reaction. In addition,both substances are manufactured by coupling pre-formed segments, i.e., by the convergent synthesisapproach. More specifically, Desmopressin is synthe-sized via a 31 41 2 segment coupling strategy, whileAtosiban is assembled by coupling the segments in a2 1 5 1 2 mode.

In contrast to the strategy applied to manufacturethe Atosiban molecule, Desmopressin drug substanceis synthesized via a linear nonapeptide, utilizingN-a-Boc-protected amino acids and the minimal side-chain protection approach. Thus it proved possible todevelop a successful Desmopressin production pro-cess by utilizing protection of the sulfhydryl groupson Cys6 and Mpa1 only. These functional groups areblocked by the acetamidomethyl (Acm group),2

which is removed, with concomitant disulfide forma-

Table II Summary of Data Obtained for thePurification of Atosiban Drug Substance

Purification Step Yield (%)HPLC Purity

(Area %)

Crude peptide 100 ;90Ion exchange .90 .96Reverse-phase HPLC .90 .98

SCHEME 3 Overview of the purification process forAtosiban drug substance


tion, by oxidation with iodine in aqueous acetic acid.This protection group strategy, in combination withsegment synthesis, using mainlyp-nitrophenyl esterand DCC/HOBt mediated couplings, yields an eco-nomical and efficient process for the manufacture ofhigh-purity Desmopressin.30–33 Furthermore, peptideintermediates produced in the Desmopressin processare more hydrophilic in nature than those obtained inthe production of Atosiban drug substance, primarilydue to the minimal protection group strategy applied.Therefore, intermediates isolated during the synthesisof Desmopressin are predominantly purified and iso-lated by precipitation, rather than via extraction with,

for example, ethyl acetate, as normally applied to theless polar segments isolated during the Atosiban pro-cess.

In the synthesis of Desmopressin drug substance,there are two additional features that are interesting tocomment on:

● The tripeptide hydrazide, Mpa(Acm)–Tyr–Phe–NHNH2, is obtained by reacting Mpa(Acm)–Tyr–OEt with H–Phe–NHNH2 in DMF/H2O in thepresence ofa-chymotrypsin. Using this ap-proach, employing enzyme-catalyzed couplingof commercially available phenylalanine hydra-zide, use of toxic hydrazine to form the hydra-zide from the peptide ester could be avoided. Inaddition, the enzyme-assisted coupling is highlyefficient, affording the tripeptide hydrazide di-

SCHEME 4 Overview of the solution synthesis of Desmopressin drug substance.

FIGURE 4 Structure of Desmopressin drug substance.


rectly from the peptide ester in a single step in90% yield.

● Crude Desmopressin peptide obtained in the ox-idative cyclization reaction is purified first bycation exchange chromatography on S-Sepha-rose FF, which has a dual function. Its primaryfunction is to increase the HPLC purity of theproduct to a level of greater than 98%, while itssecondary function is to eliminate the excessiodine used to form the disulfide bond. Thus, anadditional step to remove excess iodine at theend of the oxidation reaction (for example, bythe addition of a reducing agent such as sodiumthiosulphate or dithionite or ascorbic acid) isavoided. Finally, an additional purification bygel permeation chromatography on SephadexG-25 is carried out to enhance the purity of thepeptide to 99% or higher, prior to lyophilizationto give the acetate salt of Desmopressin.

OVERVIEW OF THE SOLID-PHASEAPPROACH

Background

From its inception, the solid-phase method has provento be an extremely useful tool that has been instru-mental in the development of numerous different pep-tides for research, and several excellent reviews havebeen published on the method.7,34,35Despite this, themethod has only been used in a limited number ofcases for the commercial manufacture of peptide drugsubstances (Table I). As noted above, the solid-phasestrategy is most frequently used to manufacture drugsubstance during the early stages of development ofpeptide pharmaceuticals, principally because little de-velopment of the method is necessary, and thereforethe drug substance required for preclinical and earlyphase clinical studies, at least, can be manufacturedrelatively rapidly. Frequently, though, the manufac-turing method is changed to the solution-phase ap-proach when larger amounts of drug substance arerequired, because, for many years, the solid-phasesynthesis approach was thought to be unsuitable forthe large-scale manufacture of peptides for reasonsthat include the following:

● the high cost of the process and its lack ofscalability;

● lack of adequate in-process controls during theassembly of the protected peptide on the resinsupport used for the synthesis; and

● the generally low purity of the final products.

The relatively high cost associated with solid-phase syntheses is attributable to the nature of thechemistry on polymers, which, traditionally, useslarge excesses of protected amino acid derivatives andreagents. The excess reagents effectively drive reac-tions to completion and reduce the formation of un-desirable side products that can occur at each step ofthe process. In addition, the volumes of the solventsused for the multiple cycles at the various steps duringthe assembly of the peptide are larger than those usedin classical, solution-phase chemistry. The originalexpectation that intensive washing of the peptide-resin after each step would be equivalent to the puri-fication methods (such as crystallization), which areused in solution-phase synthesis methods, has provento be unrealistic. While it is quite certain that suchrepeated washing cycles cannot achieve the samepurification efficiency, this lower efficiency is offset,to some extent, by the use of large excesses of sol-vents and reagents, which generally leads to highchemicalefficiencies.

With the advent of new coupling methodologiesand resins for solid-phase synthesis, the overall effi-ciency of the method has generally increased. In ad-dition, the use of more sensitive in-process monitor-ing techniques for reactions on the polymeric matrixcan further increase the efficiency of the process (seediscussion below). Furthermore, the use of reverse-phase HPLC methodology for purification of the com-plex mixtures that often result from solid-phase syn-theses, especially when used in combination with anorthogonal method, such as ion exchange chromatog-raphy, has proven to be extremely efficient, and has,for the most part, offset the concern about the purityof crude products resulting from the synthesis. There-fore, most, if not all, of the original concerns about thesuitability of the SPPS method for scale-up are nolonger valid.

General Considerations

Despite the introduction of many new protectinggroup strategies, the most common solid-phase meth-ods in use today are those based on the originalBoc/benzyl strategy, first introduced by Merrifield,2,36

and the Fmoc/t-butyl strategy.7,35Each strategy has itsown advantages and drawbacks, and during the de-velopment phase of a project, the two methods areoften compared. Regardless of which method is ulti-mately chosen, though, considerable effort must beexpended, as with any other project, on the optimiza-tion of the individual steps in the process. While theextent of optimization will depend on the project, and


particularly on the scale, the following general syn-thetic steps must be considered:

● attachment of the first protected amino acid de-rivative to the polymeric carrier;

● cleavage of the temporary protecting group;● coupling of the remaining amino acid derivatives

in the desired peptide sequence;● multiple washes of the resin after each step;● detachment of the peptide (either protected or

deprotected) from the polymeric carrier;● cleavage of the temporary and permanent pro-

tecting groups on the resin or in solution;● isolation of the crude peptide;● purification of the peptide; and● isolation of the final product.

Resin Supports.The choice of the resin for a partic-ular synthesis (i.e., type of resin, loading, handlemodification, etc.) and the chemistry used for attach-ing the first (i.e., C-terminal) amino acid in the se-quence are mainly determined by the peptide se-quence and the protecting group strategy to be used,and all of these factors can have a profound effect onthe success of a synthesis. A variety of differentpolymeric matrices are now available for use,6 with aneven greater selection of chemistries for attachment,although the original crosslinked polystyrene matrix,first proposed by Merrifield, is still the most widelyused. However, in all strategies, it is critical that themethod for attaching the first amino acid should beboth high yield and free of racemization. Furthermore,compatibility of the solvent or solvent mixtures withboth the polymeric carrier and also the protectedpeptide chain being assembled is of critical impor-tance at every step of the process, since solvation ofthe reaction centers on the polymer influences theirreactivity. Therefore, the choice of the solvent or thesolvent mixture plays an important role, particularlyin coupling steps. If a coupling is repeated, the solventmay be changed, while, in other steps (e.g., cleavageor neutralization), the reaction medium is determinedby the reaction step itself and is not normally changedsubsequently.

Cleavage of Temporary Protecting Groups.Cleav-age of temporary (i.e., N-a) protecting groups duringthe peptide assembly stage is an important step in allpeptide synthetic processes, and the orthogonalityprinciple34 should be applied, whenever possible, asin the case of the Fmoc/t-butyl strategy. The N-aprotecting groups are generally cleaved by more uni-form methods in the Fmoc/t-butyl strategy (e.g., by asolution of piperidine in DMF) than in the Boc/Bzl

strategy, where many suitable acidic reagents can beused (see discussion below). Despite its high cost andenvironmental concerns, TFA is still the most fre-quently used reagent for acidolytic removal of the Bocgroup.

Coupling Reagents.As with the solution-phase ap-proach (see discussion above), a wide range of cou-pling reagents and methods are available for use in thesolid-phase approach, and the choice is often influ-enced both by the type of the peptide sequence andalso any “difficult couplings,” which may be pre-dicted in advance.37,38 Carbodiimides [i.e. DCC andN,N-diisopropylcarbodiimide (DIC)], mediated by1-hydroxybenzotriazole, are often the preferred re-agents for this purpose, because of their low costcompared with alternate reagents, such as TBTU15

and HBTU,16 although protected NCAs (UNCAs)have been shown to be extremely effective recent-ly.18,39

Cleavage/Deprotection.In most solid-phase strate-gies, the side-chain deprotection step and detachmentfrom the resin are carried out in a single step. Themethod for detachment of the peptide from the matrixis determined by both the C-terminal modification ofthe peptide (if any) and the synthetic strategy em-ployed. Careful optimization of this step is usuallycritical, especially in the Boc/Bzl strategy, since theoutcome can have profound impact on the total yield,impurity profile, and the overall economy of the pro-cess. Both the Boc/Bzl and the Fmoc/t-butyl strategiesrequire acidic conditions for this step, and the choiceof scavengers is critical for both methods. The formerstrategy requires very strong acids, such as anhydrousHF or trifluoromethanesulfonic acid, however, whichcan present serious concerns on scale-up. On the otherhand, the milder acidolytic deprotection conditions,enhanced safety, and decreased environmental con-cerns of the Fmoc/t-butyl strategy typically can influ-ence both the development strategy and the finalchoice of manufacturing method.

Purification/Isolation. The purification of the finalproduct is, obviously, one of the most critical steps inthe process. The choice of methods is dependent onthe nature and complexity of the contaminants in thecrude product. As noted above, because of the factthat no intermediate purifications are performed in thesolid-phase method, the crude products often containcomplex mixtures of contaminants, such as deletionsequences, truncated sequences, and diastereomers,which can present a considerable challenge on puri-fication. While preparative reverse-phase HPLC can


often be used on a laboratory scale, a combination oftwo “orthogonal” methods, such as ion-exchange andreverse-phase HPLC, is often employed on a largescale (see discussion below). However, it is importantto reiterate that, since purification steps are oftendemanding of time and resources, minimization of theextent of side products through careful optimizationof all synthetic steps, as well as the cleavage step, canbe of critical importance to the overall success of alarge-scale solid-phase process (see discussion be-low).

Economic and Environmental Considerations.Asnoted above, in the discussion of the solution-phaseapproach, a number of other factors must be consid-ered when processes are scaled up. These include thefollowing:

● the economy of the process;● the purity of the final product;● the safety of the process; and● the treatment of waste and its impact on the

environment.

The economy of a process is, obviously, primarilydetermined by its efficiency (i.e., the cost of rawmaterials, the processing time, and the overall yieldand purity of the final product), and also by its ro-bustness and reliability. However, waste disposalcosts and the costs associated with complying withsafety and environmental regulations are also criticalfactors that must be taken into account during the finalchoice of the method. The Fmoc/tert-butyl strategy isgenerally considered to be more “environmentallyfriendly” than the Boc/Bzl strategy, and environmen-tal considerations may, in fact, be the determiningfactor in the event that the use of particularly hazard-ous chemicals, such as hydrogen fluoride, which isoften used in the Boc/Bzl strategy, is not permitted. Inthe absence of such considerations, both approachesare normally compared during the screening phase ofdevelopment. The final choice of method may then bemade on the basis of the total yield of peptide ob-tained in the desired quality, vs the raw material andmanufacturing costs, together with the expected totalmanufacturing requirements for the product. Whilethe raw materials for the Boc/Bzl strategy are gener-ally considered to be less expensive than those for theFmoc/t-butyl approach, the difference may be offsetby lower waste disposal costs and/or processing costsfor the latter approach.

A critical factor, which can strongly influence theyield of the final product regardless of the protectinggroup strategy used, relates to the spectrum of impu-

rities contaminating the product. Frequently, the pres-ence of contaminants, such as deletion sequences anddiastereomers, can create significant problems duringpurification, since the relative retention times of thepeaks corresponding to these contaminants are oftenvery similar to that of the main peak. Removal of suchcontaminants can therefore be very difficult, leadingto substantial reductions in yield and correspondingincreases in cost. It is thus critical to use a sensitivein-process method for monitoring coupling reactions(see discussion below), and that end-capping, for ex-ample, by acetylation, be performed in order to min-imize the formation of deletion sequences. Similarly,the coupling reagent should be chosen carefully, so asto minimize the possibility of racemization. If purifi-cation difficulties are encountered in the early devel-opment stage, however, it is may be prudent to recon-sider the synthetic approach rather than to search fora more efficient purification method.

Application of Novel Methodologies toProblems Encountered on Scale-Up

After almost forty years’ of use, solid-phase synthesison a laboratory scale is now considered to be rela-tively routine. However, when a process is first scaledup for commercial use, invariably substantial prob-lems are encountered, which can be both technical innature, as well as economic and environmental, asdiscussed above. The challenges that these problemspresent often require the development of novel solu-tions, some examples of which are discussed below.

Cleavage of Temporary Protecting Groups in theBoc/Bzl Strategy.In the Boc/Bzl strategy, in whichthe temporary (i.e. ,N-a) Boc-protecting group iscleaved under acidic conditions, there is rarely com-plete orthogonality with the conditions for removal ofpermanent (i.e., side-chain) protecting groups, whichare almost all cleaved to a small, but significant extentunder acidic condition. Under the cleavage conditionused for the Boc removal (TFA, HCl solutions), forexample, the Z and Bzl protecting groups show sig-nificant instability, and even the tosyl group, com-monly used for protection of arginine, has limitedstability. While this may not pose problems in labo-ratory-scale syntheses, even minor instabilities ofside-chain protecting groups can cause significantproblems during large-scale manufacturing, becauseof significantly longer mixing and draining times, andthe resulting formation of small amounts of side prod-ucts can complicate the validation of critical steps.

The reagent which is most commonly used for Bocremoval is 20–50% TFA in dichloromethane


(DCM),40 the excellent swelling properties of whichare a significant advantage. However, the high cost ofthis reagent, coupled with significant safety and wastedisposal issues, has prompted the investigation ofalternate, less expensive cleavage reagents.41 Theseinclude the following:

● 10% sulfuric acid in dioxane;● 0.5M methanesulfonic acid in 1:9 dioxane/

DCM;● 4 N HCl in dioxane, or ethyl acetate;● 1 N HCl in acetic acid; and● HCl in mixtures of solvents (e.g., acetic acid/

DCM, acetic acid/toluene, etc.).

While many of these reagents have found utility inthe solution-phase approach, all of them cause signif-icantly less swelling of peptide-resins than TFA-con-taining mixtures, leading to reduced accessibility tothe reaction centers. Therefore, efficient methods forin-process control are essential (see discussion be-low). This “disadvantage” can, however, be used toadvantage in large-scale processes, when largeramounts of peptide can be synthesized on highlyloaded resins in reactors of smaller volume, comparedto the classical method of assembly, in which TFAsolutions are used during the cleavage step.

HCl-containing mixtures42,43 are particularly at-tractive, because of the low cost of HCl gas and thelack of serious environmental concerns. However, thefact that such mixtures are best prepared immediatelybefore use, or even in situ, can cause problems, espe-cially from the standpoint of cGMP compliance issuesand process validation. A particularly critical factor,which must be taken into consideration with HCl-containing mixtures, is the relative rates of cleavageof the Boc group and the benzyl ester linkage to theresin, in the case of sequences containing a C-terminalcarboxylic acid. For this reason, it is particularlyimportant to monitor carefully the decrease of poly-mer loading during the assembly stage, due to thelimited stability of the benzyl ester bond toward thesereagents. A technical problem associated with the useof HCl-containing reagents—their highly corrosivenature—requires that all exposed metallic parts ofequipment must be protected by coating with an inertplastic material such as polyethylene or, preferably,by more resistant Teflon.

We have investigated the suitability of an indus-trial process using HCl in DCM, prepared in situ, as areagent for removal of the Boc group during solid-phase synthesis.44 At 15°C, it is possible to prepare anapproximately 1% solution of HCl in DCM, while atlower temperatures, the concentration increases con-

siderably. Since the rate of cleavage is determined byboth the concentration of HCl in DCM and the tem-perature, it is clearly important to control the temper-ature during the addition of the HCl gas. The reagentmay be prepared directly in the solid-phase reactor byintroducing a gentle stream of gaseous HCl, via thefritted disk at the bottom of the reactor, into thesuspension of the Boc-peptide-resin in DCM, withoutadditional cooling of the reaction mixture. The tem-perature should be maintained between 25 and 30°C,by controlling the rate of addition of the HCl gas,throughout the time of reaction (30– 60 min). If therate of addition of the HCl gas is too high, the tem-perature of the reaction mixture decreases, due toevaporation of DCM, resulting in a considerable in-crease in the HCl concentration. Despite the increasedconcentration of HCl, the rate of cleavage of the Bocgroup decreases at lower temperatures since, appar-ently, the higher concentration of HCl is less impor-tant in the first-order kinetics of this cleavage reac-tion.

Using this technique, the Boc group can be re-moved completely, without significant cleavage of thebenzyl ester linkage to the resin, throughout the se-quence of peptide assembly, and the technique hasbeen applied successfully to a number of peptides upto 10–15 amino acids in length. Since the in situsaturation of DCM by HCl does not increase theswelling of peptide-polymer during the cleavagesteps, it is possible to work with a larger quantity ofhighly loaded (1–1.3 mmol/g) resin, compared withsyntheses in which 50%TFA/DCM is used as cleav-age reagent. Furthermore, the technique is more “en-vironmentally friendly,” which makes it attractive touse for large-scale manufacturing. Further optimiza-tion is necessary, however, because of the need toeliminate water after each coupling, as well as thecontrol of temperature increases during exothermicdeprotection reactions, both of which can affect thereproducibility of the method.

Detachment of the Peptide from the Resin.Detach-ment of the peptide from the resin after assembly,often with simultaneous removal of side-chain pro-tecting groups, is a critical step, during which manyside products can be formed. Optimization of this stepis therefore critical to the yield and purity of the finalproduct and to the economy of the process. Whilemost of the peptides produced by the solid phase arethose containing either C-terminal carboxylic acid oramide groups, C-terminal esters and alcohols can alsobe important. Methods of detachment leading to thesetypes of compounds are discussed below.


Formation of C-Terminal Amides by Ammonoly-sis.36,45 Ammonolysis is used for the detachment ofmany important peptides, including neurohypophy-seal hormones and analogs, as well as analogues ofLH-RH. Traditionally, the detachment has been car-ried out by suspending the resin-peptide in methanolor in mixtures of solvents, such as DMF:MeOH 4:1 ordioxane/MeOH 9:1, to which dry gaseous ammoniawas added to give an ammonia concentration of about20%. The reaction proceeds through the methyl esterintermediate and this is also the most common sideproduct, together with the carboxylic acid, which isformed as a result of ester hydrolysis. The require-ment to keep the methanol and ammonia dry cancause problems when the technique is used for large-scale manufacturing, however.

We have studied the ammonolytic reaction in avariety of solvents in which the solubility of theprotected peptide amide varied widely. It was foundfrom these studies that the choice of solvent is ofimportance and, surprisingly, the purity of the productwas the highest in a solvent (toluene) in which thesolubility of peptide was very limited. This observa-tion, together with environmental concerns and effortsto improve the economy of the process, suggested thatgaseous ammonia, in the absence of any solvent,could be used for the ammonolysis step.46–48A sim-ilar method, using slightly different reaction condi-tions, had been described previously for multiple pep-tide synthesis applications and in the detachment ofthe libraries. In this very simple and industrially suit-able process, dry peptide-resin is placed in a pressurevessel, gaseous ammonia is introduced to a pressureof 2–5 bars, the vessel sealed, and the peptide-resinmaintained under ammonia pressure for 2–3 days.After this time, the ammonia is removed under re-duced pressure and the peptide extracted with a suit-able solvent (preferably DMF).

The results obtained for several peptides synthe-sized on Merrifield resin and cleaved by ammonolysisby this method are summarized in Table III. Theseresults demonstrate that gaseous ammonolysis is asuitable method for both the laboratory and produc-tion scale synthesis of peptide amides, which can be

used with a variety of different types of carriers and insimultaneous multiple peptide synthesis.47,48 Theyield is usually dependent on the C-terminal aminoacid in the sequence and the process must be opti-mized for each individual peptide. The method ap-pears to be relatively mild, however, and also moreefficient than the methanol-ammonia approach.

Formation of C-Terminal Carboxylic Acids by Ac-idolysis. Carboxylic acid derivatives of peptides canbe obtained by direct cleavage of peptide-resins. Inthe Boc/Bzl strategy, HF is frequently used for thispurpose, often in the so called low–high modifica-tion,49 in which a solution of HF in dimethylsulfide isused to suppress alkylation side reactions, because ofthe change of reaction mechanism from SN1 to SN2.On a large scale, however, technical problems can beencountered, because of the need for reactions to becarried out in special Teflon equipment and also be-cause HF is very toxic and highly hazardous. Substi-tution of HF by trifloromethanesulphonic acid(TFMSA) can have certain advantages.50,51 Simulta-neous deprotection and detachment has been per-formed using a cocktail consisting of an approxi-mately 10% solution of TFMSA in trifluoroacetic acidcontaining dimethylsulfide and anisole. The TFMSAmust be from a freshly opened container that has beenstored under argon.

The cleavage cocktail described above has beenused in the manufacturing of Somatostatin. The syn-thesis was performed using the Boc/Bzl strategy on aMerrifield resin, with the following protecting groups:Cys(Acm), Trp(For), Ser(Bzl), Thr(Bzl), and Lys(2-Cl-Z). The cleavage reaction was carried out on abatch of 500 g of protected peptide-resin, in a special10 L reactor, equipped with a polypropylene filter, airdriven stirrer, and an efficient cooling system. Thecleavage reaction was performed twice (for 5 and 3 h)under an inert atmosphere at 10°C. Using this method,it was possible to cleave the peptide from the resin asits C-terminal carboxylic acid in good yield and pu-rity. Isolation was accomplished by combining theacidic filtrate, with efficient cooling, directly with theDMF extract of the resin. The product was surpris-ingly stable in this very acidic mixture and it was

Table III Use of Gaseous Ammonia for the Production of Peptide Amides

Peptide Peptide-Resin (g) Detached Peptide (g) Yield (%) Purity (%)

Desmopressin 2417 1350 85 86Terlipressin 3615 2201 86 70Oxytocin 2150 1150 95 78Carbetocin 1095 510 85 82LH-RH 750 390 80 90


possible to concentrate the solution by evaporation.After redissolving the concentrate in a large volumeof DMF, the Acm groups were removed and thedisulfide bridge formed with the aid of iodine in avery rapid (15 min) reaction. After neutralization inwater–DMF, the Somatostatin was isolated by ionexchange chromatography. The procedure was repro-ducible, and could be validated without any of thetechnical problems that could be expected with theuse of HF.

Formation of C-terminal Esters by Transesterifica-tion. Detachment of the peptide from the resin as theester is a base-catalyzed reaction, for which the so-called Beyermann Reagent [1M triethylamine (TEA)in MeOH] is frequently employed. Due to low swell-ing of the peptide-resin in MeOH alone, mixtures ofsolvents are often employed, in order to improve theaccessibility of the alcohol-containing solvent mixtureto the reaction sites on the resin. Alternatively,2-(dimethylamino)-ethanol may be used as a 50%solution in DMF.52 This autocatalytic transesterifica-tion reaction, unfortunately, can be negatively influ-enced by the low stability of DMF in this mixture, andyields are often relatively low. We have found that themost efficient reagent for transesterification, which isalso applicable to industrial-scale syntheses, is a2–5% solution of 1,8-diazabicyclo[5,4,0]undec-7-enein MeOH. This method has been used to cleave pro-tected fragments of calcitonin and LH-RH in a rela-tively short time (30–60 min) with high yields (85–95%). Since the major side reaction in this type oftransesterification reaction is racemization, themethod is preferred for sequences in which the C-terminal amino acid is glycine or proline, otherwisethe extent of the racemization should be monitoredcarefully.

Formation of Peptide Alcohols by Reductive Cleav-age. Peptides containing C-terminal alcohol functionsare usually stable toward the action of exopeptidasesand can, therefore, have prolonged duration of actionsin vivo. An example of such a peptide, which ismanufactured for pharmaceutical use, is Octreotide.We have developed a synthesis for this product inwhich the protected peptide is assembled on a Merri-field resin as Boc–D-Phe–Cys(Acm)–Phe–D-Trp–Lys(2-Cl-Z)–Thr(Bzl)–Cys(Acm)–Thr(Bzl)–O–resin.Treatment of the peptide-resin with NaBH4 in a mix-ture of THF and water at elevated temperatures(50°C) for 2 h results in cleavage of the protectedpeptide as its C-terminal Thr(Bzl)-ol derivative in70–75% yield.

Monitoring of Coupling Reactions.A weakness ofthe solid-phase method in general, especially from the

point of view of large-scale manufacturing and cGMPrequirements, is in-process control during the peptideassembly stage. Since the growing peptide chain isbound to the insoluble carrier during all steps, i.e.,during deprotections and couplings, the possibilitiesto perform reliable analytical evaluation of the pro-cess, in the terms of peptide properties and state-of-the-art analytical chemistry, are very limited. Themethods used for monitoring the completeness ofcleavage of temporary protecting groups and com-pleteness of the coupling reactions can be divided intoseveral groups according to the purpose or method.Most of the tests in use currently are carried out withsmall (a few mg) disposable samples of peptide-resin,which are removed from the reactor during the assem-bly stage. Other “nondestructive” methods exist,which provide the required information by a change incolor of the entire contents of the reactor, or by thetitration either of the washings or the resin in thereactor. The presence of the free or blocked aminogroup is the control for the test and both the Boc/Bzland Fmoc/tert-butyl strategies can use the same tests.In addition, the Fmoc/t-butyl approach can take ad-vantage of the uv and fluorescence properties of theFmoc group itself and its cleavage product, dibenzo-fulvene. The same is true for the less commonly-usedDdz group. Such uv monitoring has been used incontinuous flow systems and in some automated solidphase synthesizers.

The following tests are the most frequently usedfor in-process control:

● the ninhydrin (Kaiser) test53; sensitivity: 0.02meq/g;

● the chloranil (p-tetrachlorbenzoquinone) test54,55;sensitivity: 0.02 meq/g (suitable for proline);

● the picric acid test56; sensitivity: 0.01 meq/g;● the bromphenol-blue (39,3,59,50-tetrabromosul-

phophthalein) test57,58; sensitivity: 0.015 meq/g;● the pyridine-hydrobromide test59; sensitivity:

0.02meq/g; and● self-indicating active esters (e.g. 3,4-dihydro-3-

hydroxy-4-oxo-1,2,3-bezotriazine esters ofFmoc protected amino acid derivatives)35,60;sensitivity: 0.03 meq/g.

The sensitivity of the individual tests can vary anddepend considerably on the peptide sequence, due tothe difference in the accessibility and reactivity of theamino groups on the polymer. It should be empha-sized that, for secondary amines, the ninhydrin testcan provide false results and, therefore, the chloraniltest is recommended.


Bromophenol blue is a sensitive indicator, with theability to monitor the course of couplings in a similarmanner to the self-indicating active esters of 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine.60 Bothreagents can be added to the reaction mixture after thedeprotection (neutralization) step and the resultingcolor changes monitored qualitatively or quantita-tively. However, this test can provide false results ifused during the synthesis of longer peptides, espe-cially, when basic amino acids, including histidine,are present in the sequence.

Titration of the entire content of amino groups inthe reactor with perchloric acid61 was used manyyears ago in Syn1, one of the first automatic synthe-sizers of Danish origin. However, the method has notfound widespread use because of problems with thestability of the peptide on the resin under the acidiccondition used during the titration.

Hydrobromide exchange between the weakly basicpyridine (pyridinez HBr) and the more basic primaryamino group on the resin-peptide forms the basis ofanother monitoring method.59 After neutralizationwith tertiary base, an aliquot of the washings contain-ing HBr can be titrated argentometrically to determinethe loading of the resin after deprotection steps.

We have used the picric acid test effectively in ourlaboratory to determine residual free amino groupsafter coupling reactions. Couplings were consideredas complete if less than 0.01 meq/g of free amine wasfound, although frequently this value was as low as 5eq/g. Alternatively, the same test can be run after eachdeprotection step, when decreasing substitution val-ues at each step corresponds to the increasing amountof peptide bound to the resin. Deviations from linear-

ity can be an indication of chain termination or loss ofpeptide (i.e., premature detachment of peptide fromthe resin). The test sample of the peptide-resin re-moved after the deprotection step can also be acety-lated and the treatment with deprotecting reagent andthe picric test repeated. Any change in the result is anindication of a deprotection problem.

Direct monitoring of the peptide assembly can beachieved through the detachment of the peptide fromsmall samples of the peptide-resin by ethylaminolysisor gaseous ammonolysis, both of which are rapidenough to detach small quantities of peptide which aresuitable for an in-process test using HPLC analysis ofpurity. In practice, a combination of different moni-toring approaches is recommended, especially duringthe development stage. Once the process is defined, asingle, routine test is usually sufficient and the ninhy-drin or picric tests are frequently chosen for thispurpose.

Cyclization On-Resin—An Unusual Side Reaction.Cyclization of peptides on the resin, prior to cleavage,has been proposed as a method of achieving the highdilution necessary for such a reaction, because of therelatively large distance between reaction sites onthe resin, which results in a “pseudodilution” effect.We have investigated this approach for the synthesisof Carbetocin (deamino-carba-1,2-O-methyltyrosine-oxytocin), an analogue of oxytocin used in veterinaryindications (Scheme 5). An allyl protecting group wasused in the synthesis of the cysteine derivative [N-t-butyloxycarbonyl-S-(3-allyloxycarbonylpropyl)cys-teine],62 which served as the key derivative during thesynthesis of the peptide-resin. After cleavage of the

SCHEME 5 Solid-phase synthesis of Carbetocin drug substance.


allyl and Boc groups, the carba-bridge was formedon-resin with the aid of DIC/HOBt coupling. Subse-quently, the crude carbetocin was detached from theresin by gaseous ammonolysis in high yield.

During purification, the side product,D-Asn5-car-betocin,63 was identified, although it was not clear inwhich step this diastereomeric peptide was formed.While the extent of formation of this side productdepended on the conditions used for cyclization, thiswas not the only factor. It was found that, not surpris-ingly, D-Asn5-carbetocin is formed when either theresin-peptide or the peptide in solution (during isola-tion) is exposed to basic pH (pH.7.5). Under theseconditions, the change of theL-Asn configuration wasselective and the mechanism of the reaction was notconventional racemization, but rather inversion ofconfiguration. By simply exposing carbetocin to basicpH, it was possible to obtainD-Asn5-carbetocin ingood yield. However, the reverse reaction (i.e., for-mation of carbetocin fromD-Asn5-carbetocin) was notobserved. The biological activity of this diastereo-meric peptide is known to be negligible, and allchanges in position 5 of neurohypophyseal hormonepeptides usually lead to the almost total abolition ofthe hormonal effects. This fact and the observed sen-sitivity of the Asn residue to the inversion of itsconfiguration lead us to the conclusion that similarnatural mechanisms can occur in vivo as a means ofinactivating neurohypophyseal hormones and elimi-nation of their activities.

OVERVIEW OF THE HYBRIDAPPROACH

Background

As noted above, the availability of new resins andresin handles has opened up the possibility of synthe-sizing fully protected peptide segments rapidly by thesolid-phase technique, which can be assembled sub-sequently by solid-phase64 or solution-phase9,65meth-ods. While this approach is not new, it has not beenwidely exploited, although it is used extensively as adevelopment tool for solution-phase projects, as notedabove. The approach is particularly attractive for themanufacture of large molecules, since it combines theadvantages of both the solid-phase and the solution-phase methods. In particular, the solid-phase synthesisof fragments can be developed and scaled-up rela-tively rapidly, and avoids many of the solubility prob-lems often encountered in solution-phase syntheses ofrelatively long segments. Production cycle times areshort, compared with solution-phase methodologies,

and yields and purities are often higher, because of theuse of excess reagents, especially during couplingreactions, often resulting in intermediates that do notrequire purification. Through optimization of the se-quences of the fragments, especially on the basis oftheir solubility characteristics, the final stages of theprocess, i.e., the fragment assembly and deprotection,can be scaled-up by conventional, solution-phasemethodologies. Thus, the advantages of both the sol-id-phase method, i.e., rapid synthesis of fragmentswith high purities, and also the solution-phasemethod, i.e., full monitoring of coupling reactions,with isolation, purification, if necessary, and full char-acterization of intermediates, can be exploited.

Example of the Hybrid Approach

Such a hybrid approach is being applied currently tothe synthesis of T20,66 a 36-amino acid peptide, de-rived from the ectodomain of HIV-1 gp41, whosestructure is shown in Figure 5. The product, which isthe first member of a novel class of antiretroviralagents that inhibit membrane fusion, is being devel-oped for the treatment of HIV and is currently inPhase 2 clinical trials. During the early developmentphase of the project, the product was manufactured byconventional solid-phase strategy, using the Fmocstrategy. In order to meet the projected requirementsfor metric tons of the product on commercialization,however, the strategy was changed, at a relativelyearly stage, to a three-fragment hybrid approach,which is outlined in Scheme 6.

The three fragments—(1–16), (17–26), and (27–35)—are assembled on 2-chlorotrityl resins,8 via theFmoc strategy, using HBTU-mediated couplings andthe following side-chain protecting groups: Boc onlysine and tryptophan; trityl on asparagine, glutamineand histidine; andt-butyl on serine, threonine, ty-rosine, aspartic acid, and glutamic acid. After assem-bly, the protected fragments are cleaved from theresin by multiple treatments with dilute (1%, v/v)TFA in methylene chloride, with neutralization of theTFA with pyridine. After concentration of the solu-tions, the products are isolated by precipitation in highyield and high purity and are used without furtherpurification for the solution-phase fragment assembly.

In the first step of the fragment assembly, Fmoc–(27–35)–OH is coupled with Phe–NH2, using HBTU

FIGURE 5 Structure of T20.


in the presence of HOAt, to give the (27–36) frag-ment. After removal of the N-terminal Fmoc group,H–(27–36)–NH2 is coupled with Fmoc–(17–26)–OHin the same manner. Finally, after removal of theFmoc group, H–(17–36)–NH2 is coupled with Ac–(1–16)–OH, by the same coupling technique, to give thefully protected final molecule. Deprotection is accom-plished using a “cocktail” containing TFA, dithiothre-itol, and water to give the final crude product. In allcases, intermediates are isolated by simple precipita-tion techniques and purified by recrystallization ortrituration. The yields of all reactions are uniformlyhigh and the HPLC purity of the final crude product is.70%. The crude product is purified by preparative,reverse-phase HPLC techniques and isolated by ly-ophilization.

The synthesis of T20 is a rather remarkable exam-ple of the application of modern peptide synthesistechniques to a very challenging scale-up problem.Even though the process has not yet been scaled upfully, T20 is already being manufactured in batches of

.10 kg. It is certainly the most complex peptide to bemanufactured on such a large scale, and based on thelevel of success achieved in the project to date, it islikely that the ultimate goal of the project will beachieved, i.e., manufacture of metric tons of the prod-uct on an annual basis.

PURIFICATION AND ISOLATIONTECHNIQUES

While optimization of every step in the manufactureof a peptide drug substance is important, in order toensure that the product can be manufactured as eco-nomically and reproducibly as possible, the final stepsin the process, i.e., purification and isolation, arecritical to any process, regardless of the syntheticapproach used. In principle, a variety of methods ispotentially available for the purification of peptidedrug substances, of which the following have histor-ically been considered:

SCHEME 6 Outline of the synthesis of T20 drug substance.


● crystallization;● countercurrent distribution;● partition chromatography;● gel permeation chromatography;● low-pressure, hydrophobic interaction chroma-

tography;● ion exchange chromatography; and● reverse-phase, high performance liquid chroma-

tography.

Since peptides are, in general, difficult to crystal-lize, crystallization is rarely attempted for final puri-fication steps, except in the case of small (i.e., fiveamino acids or less) molecules. While countercurrentdistribution and partition chromatography were usedfor purification of many of the early peptide drugsubstances, they have largely been replaced by morepowerful techniques, such as those based on prepar-ative, reverse-phase HPLC.

Today, therefore, in contrast to most other drugs,pharmaceutical peptides are normally isolated andpurified by chromatographic procedures, which aremore or less mandatory, due to the complexity of theproducts and the strict purity demands from regula-tory agencies. The most common chromatographictechniques in use to today are gel permeation, lowpressure hydrophobic, ion exchange and reverse-phase HPLC chromatographies. Obviously, wideranges of chromatographic media are available foreach type of chromatography, and the choice of thecombination of purification steps and individual me-dia used will be determined by the nature of thepeptide and impurities it contains. Since chromato-graphic procedures, in general, are demanding of timeand resources, the purification of the final productshould be performed in as few steps as possible.Ideally, a single purification step should be utilized,since every chromatographic step potentially de-creases the total yield, although this is often difficultto achieve in practice. In a typical purificationscheme, the crude product from the synthesis is firstsubjected to a step—such as ion exchange, gel per-meation, or hydrophobic interaction chromatogra-phy—which is designed to remove by-products fromthe final deprotection step(s), most of which are lowmolecular weight and uncharged, and may also affordsubstantial purification of the product. If further pu-rification is required, a final “polishing” step may beperformed using a complementary technique, such asreverse-phase HPLC. Finally, the organic modifierfrom the reverse-phase HPLC step (e.g., ethanol oracetonitrile) is evaporated and the product isolated bylyophilization, which, preferably, provides the finalproduct in the correct salt form (typically acetate)

without the need for a separate step. Such a purifica-tion scheme, using two complementary techniques, isoften more efficient than, for example, the use ofseveral reverse-phase HPLC steps in different mobilephases.

Chromatographic purification and lyophilizationare both expensive steps, which, together, may actu-ally contribute the major part of the cost of manufac-turing a particular peptide on a large scale. Further-more, limitations may be encountered, particularlywith reverse-phase HPLC purification and lyophiliza-tion steps, when manufacturing on a very large scale(i.e., metric tons). In such cases, it is, obviously,important to optimize the process, if possible, to thepoint where the requirements of the final purificationstep are less demanding. If this can be achieved,techniques such as ion exchange chromatographyand/or low-pressure hydrophobic interaction chroma-tography, both of which can be scaled-up with fewlimitations, may be adequate for production of thefinal product at the required level of purity. Further-more, alternate techniques for final isolation of theproduct must be considered in order to accommodatethe needs for larger-scale manufacturing. In this con-text, diafiltration/ultrafiltration may be considered as auseful adjunct to any final isolation step, since itprovides a simple and scalable method of removinglow molecular weight salts and organic solvents, aswell as concentrating solutions prior to, for example,lyophilization. Alternate methods of isolation, such asspray drying or precipitation, which are more easilyscaled-up than lyophilization, should also be consid-ered.

REGULATORY CONSIDERATIONS

The ultimate manufacturing goal for any peptide phar-maceutical is the development of a process that is botheconomical and also meets the requirements of theregulatory authorities. The latter consideration is ev-ery bit as crucial as the technical aspects of the pro-cess itself, since failure to meet regulatory require-ments can result in the failure to obtain approval tomarket the product. The ability to meet the expecta-tions of the authorities requires similar planning tothat required for the technical phases of the project, inthat a “quality system” of procedures, documentation,and test methods for raw materials and finished prod-ucts, must be established that guarantees that theprocess is highly reproducible and will reliably resultin final product of consistent quality. This can be aparticularly difficult challenge for manufacturing pro-


cesses for peptides, because of the large number ofoften complex steps involved in their production.

Synthetic procedures intended for use in industrialscale production must be reliable in several ways. Asynthesis for large-scale purposes is developed in alaboratory by skilled chemists. However, it is mostlikely reproduced routinely in an environment withpersonnel less competent in chemistry but with higherquality and safety demands. Thus, the toleranceagainst variations in reaction parameters, such as tem-perature, reaction time, or reagent amounts, must beknown and acceptably high. Ultimately, the process—including the in-process analytical methods, and thosefor testing and release of the finished product—mustundergo formal validation, in order to confirm itsreproducibility, both in terms of yields of intermedi-ates, the yield of the final product and the consistencyof its impurity profile.67

Prior to approval of a product for sale, the manu-facturer of the bulk drug substance must be subjectedto an inspection by one or more of the regulatoryagencies, such as the U.S. Food and Drug Adminis-tration, for compliance with the applicable regula-tions.68 At that time, the documentation of virtuallyevery single aspect of the company’s operations, fromthe ordering procedures for raw materials, to the finalshipment of the finished bulk drug substance, must beable to stand up to in-depth scrutiny. Preparing forsuch a “Pre-Approval Inspection” can require consid-erable effort and expense on the part of the manufac-ture, but such preparation should be a part of theplanning virtually from the beginning of the technicaldevelopment phase of the project. While the overalleffort to meet the final goal of approval is still thesame, proper planning can assist by spreading theeffort over most of the life of the project. The positiveaspect of these regulatory requirements, of course, isthat the safety of the final customer, i.e., the patient, isvirtually guaranteed, because of the requirements forconsistency of the manufacturing process, coupledwith complete traceability from raw materials throughthe finished product.

CONCLUSIONS

It is clear that, almost 50 years since the first pub-lished synthesis of a biologically active peptide, by duVigneaud and co-workers, the technology now existsto manufacture complex peptides on an extremelylarge scale. Furthermore, recent advances in the areaof drug delivery have provided practical solutions tothe problem of lack of oral bioavailability thatplagued peptides for may years, and limited their use

as pharmaceuticals mostly to life-threatening dis-eases. As a result, there has been a resurgence ofinterest in peptides for therapeutic applications, whichis likely to result in numerous new peptides reachingthe marketplace as drugs. This increased demand forproduction of peptides as pharmaceuticals will cer-tainly provide a considerable manufacturing chal-lenge. However, based on the current status of thetechnology, it is likely that this challenge will be met.

The authors wish to acknowledge Brian Bray, PaulFriedrich, M. C. Kang, Maynard Lichty, Catherine Mader,and Gene Merutka of Trimeris Incorporated for their devel-opment of the manufacturing process for T20.

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