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Dr Jyothi Thundimadathil of CPC Scientificasks how peptide API manufacturers can address growing volumedemands

Peptides, which are small proteins typicallycontaining a chain of up to about 50 aminoacids, have long been seen as promisingtherapeutic agents. Over 60 are currently on themarket and are generating sale of more than $13billion/year, over 1.5% of total drug sales. 1 Four -Glatiramer, Leuprolide, Goserelin and Octreotide -have already crossed the $1 billion/year mark.Table 1 shows some of the top selling peptidedrugs.

The number of peptide drugs entering clinical

trials is increasing steadily: it was 1.2/year in the1970s, 4.6 in the 1980s, 9.7 in the 1990s and16.8 in 2000s. Currently about 140 peptides arein clinical trials and more than 400 in pre-clinicdevelopment (Figure 1). From 2000 onwards,peptides entering clinical study were mostfrequently for cancer (18%) and metabolicdisorders (17%). 2 More than 100 pharmaceuticaland biotech companies are now pursuing peptidedrug discovery programmes.

There are several reasons for the tremendousincrease of interest in peptide-based therapeutics.The most important is the major advances in drugdelivery systems for peptides in the last ten years.Certain peptides can also pass through cellmembranes and therefore can be used as carriersfor targeted drug delivery. The use of peptides isalso growing in medical diagnostics,nutraceuticals, anti-microbials and cosmetics.

The CMO sector is under tremendous pressureto meet the current need for large quantities -about 1,400 kg/year - of peptide APIs. Of thistotal, about 40% is produced by CMOs and theirshare is expected to rise drastically by 2017, whena number of peptide drugs become generic and

more peptides are approved. Moreover,pharmaceuticals companies are expected to relymore on CMOs for large-scale manufacture ofpeptides in an attempt to cut production costs.

Therefore, it is time to focus on improvingcurrent manufacturing methods in order toincrease productivity without compromisingquality. Big peptide CMOs are upgrading theircapabilities by acquiring large-scalemanufacturing equipment, strategic positioningof their facilities around the world and innovative

manufacturing methods.

Peptide manufacturingA number of chemistries and methods have beendeveloped for building peptide chains. Theclassical method, liquid-phase peptide synthesis(LPPS), involves reactions in solution usingprotected amino acids and requires the isolationof product after each coupling and de-protectionstage from solution.

Solid-phase peptide synthesis (SPPS) relies onassembling peptide chain on an insolublepolymeric support, thereby simplifying theprocess. Protected amino acids are added one byone to the resin beads after each de-protectionstep and require no intermediate isolation. Whenthe desired length of sequence is synthesised onthe resin, the peptide is cleaved off andsubsequently purified using chromatographictechniques. A mixed - hybrid or convergent -strategy involving both LPPS and SPPS also is usedin the manufacture of certain peptides.

Obviously, shorter chain (

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using hydrophobic interactions is seen as the mostpowerful method for peptide purification. It ischaracterised by the use of a stationary phase andan aqueous mobile phase containing an organicsolvent, such as acetonitrile or an alcohol.

Various chromatography media have been usedfor large-scale purifications of peptides on reversephase resins. Among the most popular are thosebased on C

4, C

8and C

18alkyl chains attached to

a silica surface.Nowadays, with the popularisation of dynamic

axial compression (DAC) columns forchromatography and the availability of excellent,relatively affordable reversed phases, thepurification of peptides has become easier andmore accessible than ever before. A 20 cmcolumn can yield several kgs/month of purifiedpeptide.

After purification, peptides are isolated anddried using lyophilisers. As well as bottlelyophilisers, peptide manufacturers use largecommercial-scale tray lyophilisers to increase

productivity and improve control of dryingparameters.

Challenges in manufacturingPeptide manufacturing technology has maturedand major manufacturers can now producepeptides APIs in hundreds of kilograms scale.With more peptide drugs poised to enter themarket and many more in trials, pressure ismounting on CMOs to offer peptides moreeconomically while still adhering to GMP andwithout compromising quality and integrity.Moreover, the complexity of peptides, in terms ofsize, modifications, conjugation methods, stabilityand purity requirements, is expected to increase.

This scenario is a challenging prospect formanufacturers. From a synthetic point of view,the resins currently being used for SPPS are notre-used and are expensive compared to other rawmaterials.

Although the prices of common amino acidsprotected with Fmoc group has come down inrecent years, many of the D-amino acid andunnatural amino acid derivatives used as buildingblocks will increase product cost. Large quantitiesof resins (only 0.5-1.0 mmol of reaction sites areavailable per gram of resin) and excesses of amino

acids (2 to 3 equivalents) are used in peptidesynthesis.

Solvents such as dichloromethane anddimethylformamide are used in large excess forresin swelling and washing steps, therebyproducing a considerable amount of waste.Typically, a minimum of 5 ml solvent/gram of resinis needed for each washing step, which meansthat when the synthesis of a 20 amino acid isperformed on a 1 kg of resin about 1,000 litres ofsolvent waste are generated.

Waste disposal and solvents amount toapproximately 10% of the total production cost.At the end of the synthesis, the peptide is cleavedfrom the resin using trifluoroacetic acid followedby precipitation with large excess of ether, furtherincreasing the amount of waste. The purity ofthe synthesised peptide largely depends on theefficiency of synthetic process, its own intrinsic

nature and the quality of raw materials used.Incomplete coupling and de-protection duringsynthesis will lead to deleted sequences andimpurities.

Purification is the most critical step in themanufacturing process. Poor solubility and theaggregation of certain peptides could createproblems during purification, so carefulconsideration of peptide properties beforepurification is crucial. Peptide modifications likecyclisation and disulfide bond formation areaccomplished before purification.

Diluted (1 mg peptide/ml) solutions of peptideafter such processing make the purification stepmore difficult, not only because of the hugevolume of solution to be pumped to the HPLCcolumn but also due to the presence of polymericmaterials formed via oxidation. Removing close-running impurities from the peptide of interestoften requires multiple purification steps usingdifferent buffer system and processing.

Purification accounts for more than 20% of theproduction cost of peptides. The price of solvents,such as acetonitrile for RP-HPLC purification,often fluctuates, further increasing purificationcosts. These solvents are generally not recoveredor reused. The freezing and drying of largequantities of peptide solution after purificationadds considerable further cost.

The manufacture of peptide APIs requires fullcompliance with GMP regulations and FDA

guidelines. Raw materials have to be qualified andall manufacturing takes place in controlled areas.Purification and lyophilisation are performed inClass 100,000 clean room areas followed bypackaging in Class 10,000 clean rooms.

Updating SPPSFigure 2 shows a process diagram for thesynthesis of peptides by SPPS. No majortechnology has overtaken SPPS since Merrifieldfirst introduced it, mainly because of its ability tosynthesise long chain peptides conveniently inhigh yield.

SPPS is quicker than conventional step-by-stepsynthesis and can be fully or semi-automated. Inlarge-scale peptide manufacturing, it allows semi-automation for solvent delivery, coupling reactionand waste management, whereas LPPS is limitedto the synthesis of shorter peptides and takes alonger time for production.

Other technologies, such as fermentation andrecombinant methods, have been introduced

recently in the manufacture of peptides such ascubicin and liraglutide. These methods can easilybe scaled up to produce large quantities, but theirwider use is limited by such issues as the use ofspecific enzymes in fermentation and the difficultyin introducing unnatural amino acids viarecombinant expression.

Improving SPPS and LPPS technology isnecessary to achieve low-cost manufacturing ofpeptides. New methods for reducing wastegeneration and improving coupling efficiency andoverall yield will drive the process costs down.

The recycling of solvents is a viable optionduring large-scale synthetic processes. The initialinvestment in a distillation plant may be high, butonce it is set up the system can be used toregenerate solvents, which will reduce wastedisposal costs as well. Efficient supply chainmanagement and consistent quality of rawmaterials from trusted vendors is necessary inlarge-scale manufacturing.

Overall, the synthetic process for peptides canbe improved by using high quality raw materials,careful management and recycling of solvents,and using efficient coupling agents andconvergent strategies. For example, a difficultpeptide with more than 50 amino acids can be

Peptides & proteins

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Pre-clinical ~400 Phase II ~75

A p p r o

v e d ~ 6

0 P h

a s e

I ~

4 0

P h

a s e

I I I

~ 3 0

Figure 1 - Peptide market by clinical stage

SPPS reactor, 20 cm RP-HPLC column and 200 litre tray lyophiliser at CPCs GMP peptidemanufacturing unit

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synthesised by fragment condensation in the solid

phase, thereby achieving better yields and puritycompared to step-wise SPPS method.

For industrial-scale purifications on reversephase resin columns, the shape and size of theparticles in the stationary phase are importantconsiderations. Spherical particles are preferred toirregularly shaped ones. Extensive use of columnsunder DAC sometime results in the formation ofirregularly shaped particles.

Particle size is another important characteristicof bonded silica phases that strongly influencescolumn efficiency. It is important to check thecolumns periodically for efficiency when they arein continuous use for a specific project.

RP-HPLC is an efficient but expensive procedurefor the purification of peptides. It is good idea tosubject crude synthetic peptide to a capturingstep in which most of the impurities are removed.This can be achieved, for example, by applyingthe crude peptide solution to an ion exchangecolumn. A fraction highly enriched with thedesired peptide is obtained after this initialpurification removing, for instance, those by-products generated in the final de-protection ofthe peptide.

This strategy is very economical for the pre-purification of a dilute solution (~1 mg/ml)

containing disulfide-bonded peptide that hasbeen subjected to oxidative folding reactions. Theinitial pre-purification will remove polymericmaterials and many other impurities, enabling aconcentrated solution of peptide to be loadedonto a RP-HPLC column for a second purification.For example, using this method, a 200 litresolution obtained after oxidation with a purity of30% can be loaded onto an ion exchange (IEX)column to get a 20 peptide solution with over75% purity.

No organic solvents are used in IEX processesand the overall purification cost for thistechnology is lower than using two RP-HPLCpurification steps. Polymeric materials andunwanted impurities can also ruin RP-HPLCcolumns if loaded directly without pre-purification. It is also easier to clean andregenerate IEX columns than RP-HPLC columns.

Another way to cut production costs is the

recycling of acetonitrile. The suitability of othercheaper alternatives, such as methanol orisopropanol, could also be evaluated in certaincases. Lyophilisation costs could be reduced byconcentrating the peptide solution collected frompurification columns by evaporation, the elutionat higher concentration of organic eluent ormembrane filtration techniques.

Updating LPPSIn recent years researchers have been exploringvarious methods to improve peptide synthesistechnology. In a modified LPPS strategy, aprocedure involving the coupling of amino acidsand peptide acids, instead of the usual aminoesters and peptide esters, to slight excesses ofpentafluorophenyl active esters in a THF/watersolvent mixture has been examined.

Due to their poor solubility, peptide acidintermediates are easily isolated in high purity byacidification under controlled conditions and theremoval of excess active esters by selectiveextraction. Unlike classical repetitive solution-phase procedures, this approach does notrequire time-consuming neutralisation reactionsand reduces significantly the number ofoperation units needed to obtain peptide

intermediates. 3Another study uses a new technology

platform called membrane-enhanced peptidesynthesis (MEPS), which advantageouslycombines organic solvent nanofiltration withLPPS. A first amino acid is linked to a solublepolyethylene glycol anchor. Through subsequentrepeated coupling and deprotection steps, thepeptide is extended to the desired length. Theresidual by-products and excess reagents aftereach reaction are removed by diafiltrationthrough a solvent-stable membrane, whichretains the peptide. 4

The purity of the peptides produced by MEPS isreportedly higher than that of those produced bySPPS, under the same conditions. MEPS benefitsfrom the advantages of LPPS, while avoiding thepurification steps that have until now made itpractically difficult.

A group of Japanese researchers has designedan efficient method for the synthesis of peptidesbearing an amide at the C-terminal. This methodinvolves the attachment of a C-terminalprotecting group bearing long aliphatic chains,followed by the repetition of simple reaction andprecipitation steps with the combined advantagesof LPPS and SPPS. Using this method, ahydrophobic peptide was successfully synthesisedin good yield and high purity, which cannot beobtained satisfactorily by SPPS. 5

Very recently a soluble tag-assisted LPPS wassuccessfully established, based on simplehydrophobic benzyl alcohols, which can easily beprepared from naturally abundant materials.Excellent precipitation yields were obtained ateach step, combining the best properties of LPPSand SPPS. This approach can also be appliedefficiently to fragment coupling, allowing thechemical synthesis of several bioactive peptides. 6

Conclusions & outlook

With increasing demand for large quantities ofpeptides and their greater complexity in terms ofsize, modifications and purity requirements, it isnecessary to look for better ways ofmanufacturing peptides. Further advances andimprovement in synthetic peptide chemistry, solidsupport materials, purification technology andisolation methods are needed to tackle currentchallenges in the large-scale manufacturing ofpeptides, especially high production costs andcomplex modifications.

Major CMOs have already started focusing onthese areas in an effort to supply highly activepeptide APIs in larger quantities needed forfuture needs at affordable prices. Properinfrastructure development is also needed tomeet future demands for several thousand kilosof peptides.

Strategically placing manufacturing unitsaround the world adhering to FDA and GMPguidelines a viable way of cutting downmanufacturing costs without compromisingquality and regulatory guidelines. The availabilityof high quality raw materials in bulk at reducedprices in these regions as well as highly qualifiedworkforce is crucial for successful operation ofpeptide manufacturing units in these regions.

Dr Jyothi Thundimadathil

DirectorCPC Scientific, Inc.Tel: +1 408 734 3800 x 110E-mail: jyothi@cpcscientific.comWebsite: www.cpcscientific.com

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Contact

1. A.M. Thayer,Chem. Eng. News2011 , 89, 13-202. Peptide Therapeutics Foundation, Development Trendsfor Peptide Therapeutics Report, San Diego,20103. C. Meneses et al., J. Org. Chem.2010 , 75 (3), 564-5694. S. So et al., Org. Process Res. Dev.2010 , 14 (6), 1313-13255. D. Takahashiet al., Org. Lett.2012 , 14 (17), 4514-45176. Y. Okadaet al., J. Org. Chem.2013 , 78 (2), 320-327

References

Label issuance &preparation

Raw materialqualification

Final QC testing QAreview & release

Equipment &area preparation

In processtest

In processtest

In processtest

In processtest

Purification of the peptide:100K clean room

Synthesised peptide detached fromthe resin

Lyophilisation of the purifiedpeptide: 100K clean room

Move to class 10K clean roompackaging room

Packaging& labelling

Sequential additional ofprotected amino acids

Attachment of first aminoacid to resin

Figure 2 - Typical steps and procedures using SPPS technology at CPC