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Title:
PROSTATIC ACID PHOSPHATASE, COMPOSITIONS COMPRISING THE SAME, AND METHODS FOR PRODUCING AND/OR PURIFYING THE SAME
Document Type and Number:
WIPO Patent Application WO/2015/102650
Kind Code:
A1
Abstract:
The present invention relates generally to prostatic acid phosphatase (PAP), compositions comprising the same, and methods for producing and/or purifying the same.

Inventors:
MITSCHELEN JONATHAN (US)
LIGHTHOLDER JOHN (US)
BREAM GARY (US)
FREEMAN KENNETH (US)
Application Number:
PCT/US2014/012029
Publication Date:
July 09, 2015
Filing Date:
January 17, 2014
Export Citation:
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Assignee:
AERIAL BIOPHARMA LLC (US)
International Classes:
C12N9/16; A61K38/46; A61P25/04; B01D15/32; B01D15/36; C12N5/07
Foreign References:
SU1576564A11990-07-07
US20100266569A12010-10-21
Other References:
LAM K. W. ET AL.: "« Biochemical Properties of Human Prostatic Acid Phosphatase»", CLIN. CHEM., vol. 19, no. 5, 1973, pages 483 - 487
XINDU G. ET AL.: "«Liquid chromatography of recombinant proteins and protein drugs»", JOURNAL OF CHROMATOGRAPHY B, vol. 866, no. 1-2, 2008, pages 133 - 153
GASSER F. ET AL.: "«Long-term multiplication of the chinese hamster ovary (CHO) cell line in a serum-free medium».", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY, vol. 21, no. 10, 1995, pages 588 - 592
ZANG M. ET AL.: "«Production of recombinant proteins in Chinese Hamster Ovary cells using a protein-free cell culture medium».", BIOTECHNOLOGY, vol. 13, no. 4, 1995, pages 389 - 392
Attorney, Agent or Firm:
BOBAY, Erin Regel (P.A.P.O. Box 3742, Raleigh North Carolina, US)
Download PDF:
Claims:
TEAT H!CH iS CLAIMED IS:

1. A method for purifying prostatic acid phosphatase (PAP) from a mixtnrs, comprising

subjecting a ste s selected from anion exchange chromatography, cation exchange chromatography,, mixed-mode c¼op¾ato.gfap'hy; and hy&ophobse interactio eb omatography, (hereby purifying MP.

2. T e method of Giaim 1 ,. wherein the two or mote- chromatography steps Emprise, anion exchange chromatography, cation exchange chromatography, i ed^mode chromatography, and hydrophobic im'emoi o elixoniatography.

3. The method " any one of Claims 1 ~2 wherei the purifying ste comprises epstact g the mixiare with an anion exchange chromatography medium;

•elating: at' least a portion of the PAiP ironx the anie« exohang chrojmategraphy medium to obtain a first PAP sampls*.

sontaedng at least a portion of the first PAP sample wi th .a cation exchange chromatography rae katt.;

eitithig at least a portion of ie PAP from the cation exchangechro ato ra hy: medium to obtain a second PAP sample;

contacting least a portion of the second PAP sample with a mixed-mode chramaiography medhn

elating at least a portion of the PAP from the ixe -mode cinematography edinm to obtain a third PAP sample:

contacting at least a portion of the hird PAP sample with a hydrophobic nisa-aodon shromato:gia l3: xr &hmi and

eliding PAP from- the hydrophobic interaction chromatography me ium to obtain a fourth PAP sample,

4. 'The method of any one of Claims ! -3; wherein ¾e anion exchange

chromatography medium and/or cation exchange chromatography medium comprise a hydrophiiie polyvinyiet er polyme ; S;

chromatography med um comprises a strosg anion exchange functional group.

& Tise method of any one of Ga bas 3-5, wherein the cation e c an e

chromato ra hy medium compxises a strong cation exchange fowrtiosaaJ group.

7. : The method of any one- f Claims l-£s, wherein the mixed-mode chromatography medi m comprises a &&r m hydyoxyapatite i xed-mode resin.

8, The method f any oi*e of Claims |-7} w erein the mixed-mode chromatography medium comprises on^ ormoes lome groups selected from Ca *> P<¾¾"* OH', and any •combination, thereof,

9 , the method of any om of Claims 1-8, wlMrei the hy dropho bic interaction chr¾nmtogfi¾5 y medium comprises am ry! functional grous.. 1 -9, farther comprising nherhtg the ptfrilied

11 , rOse method1 of Claim 10? be¾½ the isltmfihraiiGitfdiafiihatiorj ste is repeated t o or roo times.

12. The method of any am at C ms tO- i t erein $te ulira¾iir h 'd¾fiiifet io»- step Is carried oat with; regenerated celMose memiaraoe filter, -a- .modified. polyethsrsMlibue filter, andior a I drophillo polyvinyhden fluoride filter.,

O i The method of any one. of Gaft¾s: i -12, wherein the two or more cteoroatogiaphy steps are column chroKiatography steps,

14. The method ef any one of Claims 1 -13, wherein the PAP is reoorabmajsiFAP, optionally obtained from a raamrnaliaji ceil,.

1 . The method of Claim Ϊ4. wherein the recomhmaritPAP is expressed by ceil in ispitute, tiK eotere ba iug PAP titers of at least 200 mg/i,. 16, The method of srsy ooe ofCla¾∞s 1 --15, wherein, ike purified PAP as a ptrrity of reater than 95%.

I' . The- method of any -one qfCkrras 1 -16, here n the purified PAP has a purity of greater than 98%.

1 S. The rnethod of y one of Claims 1-17, u¾e.rei« the pmified P AP has a host cell, protein conten of.kso- thsttabout 100 r¾g/mg PAP,

19-, The method of any one of Claims 1-1 S, lierein tbe 'purified PAP has a imst eeP DNA corneal of less tham about TOO pg/mg AP,

20, The in.ethod' of any one of Claims I- l¾.-whe!¾>|i -l¾s,i¾.eilio ro ides m overall rec very of PAP of about 30% to aboMS0%.

2L, The method of any one of C!ate 1-20, w¾¾rein the pacified P AP has m ac ivity of at least about 250 to 350 units/tag PAP.

22. The rnethod of any one of Claims 1-21 whersin the mixture comprises cell eu tiire raedi rm

2X The method of say one of Claims 1-22, whare the PAP is secretory PAP ana t e PAP is optioRally soiiihie,

24, An isolated prostatic acid phosphatase (PAP) having a parity of greater than 60%.

25, The isolated: PAP of Claim 24, wherein the PAP has a purity of greater tha» 75%.

26,. The isolated PAP of y on© of Claims 24 or 25, wherein the PAP has a host cell protein conte of less than al t 100 ng mg P AP,

27, The isolated PAP of any ODS of Claims 24-:26; ^hsrei ¾e PAP lias a Host eel! DNA eonte of less than ahoai 100 pg/mg PAP., 28, The isolated PAP of any one of Claims 24-27, wherein the PAP

PAP. optioraaliy o tained from a snaromaiiaxi cell, PAP

30·. The coiiiposirion of Claim 29, wherein the .PA hats a parit of greater than 75¾ prior to additien to the composition.

31. The com oskio of any one of Claims 29 or 3% . wherein the PAP has a host cell proi ifi content f loss than, abaat fi}0 ng/rng PAP,

¾ ¾e CQffip&siiion of any ope of Claims 29-31, hereip the PAP has a host ceil 'DMA t t f less than .about 1M pg/mg PAP.

33, The composition, of any one of Cl ims 296T, whereirn; th? PAp is ecombinant PAP, op ionally obtained from a mammalian call,

34, A pharmaceutical '■composition comprising a pharmaceutically acceptable carrier and prostatic ae p os hatase (PAP)S wherein the PA has a pwrity of g¾¾ter than 6 ¾ priof to .addition to the pharmaceutical composition.

3& ¾e pharmaceutical cotnposition © f Claim 34, Beteio the PAP has a parity of greater thaa 75¾ prisr to addition to the pharmaceutinal. composition,

36. The p! maesiitieai eornpositiors of aay mm of Claims 34 or 35, wherein the PAP i $fcoet cell protein content of less tharn:¾botit iOOaagAng AP,

37. H pharoiaeeatica! composition af any one of Claims 34-36., wherein: the PAP has a host ell DMA content of less than a!¾ut 100 pg/srog.PAP.

38. The pharrriaeeaiicai Poraposition of asy one of Claims 34-37, wherein fee PAP is reeorahinarii AB, optionally obtairied from a. mammalian .cell A medicamerit com osing the isolated PAP of a.oy am of Claims 34-3 $ arid a p¾a£roaceuticiaily acceptable earner;

40 A method of cufiuring raaiss alian sails .e pces'saig a recoifi iiiarit prostatic acid phosphatase {PAP¾ comprising:

©oMurixsg .the mammal an cells i» a defined senan-ifee base medium di ng a growth phase; &

maiii aiamg thg:m^im¾Bai3:-ceils: m the base ¾ed¾m dnring a.: production phase by :Si¾)pfe«iefiti«g. the. base meditaa. with ,a dftfad $erui¾~iiee feed medi«?¾.

•'41·. The itiCiho of Claim 40s. tvhetein the SBanim han ceils ©oraprise Chhiese Hester Ovary (CHO ¾eIls,

* > The method of aa one of Claims 4©-42, wherem t e: base niedinro aad feeS tttsdiuin are animal component Bm

44, The method of any ene of M 40- 3* wherein the mammalian ceils are matotame&ai a tempe ure of .about 3?¾ a pH of ab sst 7,0. mi a ffissoiwd oxygen content of at least -about 50%,.

45, The method "Ό£ my m of Claims 0-44, wherein trie supplementing ste . carried out at a ceil density of at least abom 6:;d x 10*4

46, The method of an one of Claims 40-43, wherein the supplementing step Is repeated at least sk times and is carried sat ewer other day,

47, The method of any one of Claims iM6y wherein the supplementin s e :

com rises adding he feed medium as a bolus at a rate of about \ Q% pf the ir fial ewhure ums per feed ddithim 4f. The method of'any one ef Claims 40-47, wherein ihe mainiaining step comprises s im lementing t¾e base dium wit glucose.

49, 1 lie method of Claim 48, wherein glucose added to naamtain a. glucose con eniratlofi of about 3 g fa about 6 g of glucose per L.

50< The method of sy ovie ef Claims 40 ¾ herem &e ciUture has PAP titers of at least 200 lag L.

5L Tie method, of any ewe of Claims 4O-S0, hefeics tfce cultur has I'AP titers of about 200 mg/L to about 300 mg/L,

Description:
PrtiStatie M phosp atase, e »rjros¾£a»s: comprising he same f .a»i! aiet oils for

•produckg and:/o.r fia fym ibe same

This apphcMkm elauns the benefit of and pior!xy to U.S.. Patent Applicatio Serial No, I4/146 it¾ filed on January , 20.14, the eorttents of which are ' he e y ' Incorporated by reference as if recited in full herein.

A Sec eoee Listing; iu ASCII i format, submitted under 3? GJP L § 1.821 s entitled 79g-15 . ST25,.ki 5 5,334 bytes in ske f generated December 2.3, M mm filed via BFS- Web, is provided ii_ lieu of a paper copy . This Sequence " Listing is hereby incorporated by reference into ttie specification for its disclosures.

M M &i

Ibosiaiie acid phos h tase^ (PAP) is ap enzyme in the bisiidise acid phosphatase femi that ean hytbolyze phosphnmsraGes ers, such as 5*-a«cleostde niooophosphat&s.. There are both secretory arid transmembrane harms of $AP ? which can be prodaced fey the ptosta e. Human PAP m a honsodi er with a molecular weight of about 100 kDa, Research has demonstrated that PA? can serve as a market for prostate cancer and can be used to treat pain, such as chrofife pais.

The present weut!on addresses previous shortcomings in the art b rovidin PAP, coirr ositiofis comprising the same, and methods for producing and/or p¾«¾drig PAP,

A first aspect of the present invention is directed ' to prostatic acid phosphatase. (PAP) having a purity of greater than 60%.. In one embodiment the purity of the PAP provided herein may be greater than greater than § 5% > or even greater than. 98%. Purity, in one embod ment,, is measured by gel elsctropberesfs. The PAP may be isolated according to some embodiments of th present inveptiop, In one eaibodi em, t¾e PA may be isolated from a host cell The PAP may have a host, celt protein content of less than about 100 ng host eel! protein, per mg PAP and/or a host ceil DMA content of less than about iOO pg lost ceil DNA pe tag PAP. The PAP, in o embodiment may be teaorribinaut PAP:, optionally

-I- o iai&e ir ra a anmsatiamcelL for example, a wnm cell, The PAP ,may be obtained, iirffiedj midf produced according to a method of the tecent ventfon.

A seemed aspect of the present inventio is direeted to compositloaS j pkatmaceuti-eai compositions, and medicaments including PA? according to embodiments- -of the .present invention, 1¾e P P m y be obtained, pariSed, and/or produced according: to a method of the present invemiom

Another aspect of the present invention is directed to a. method of treating a subjec comprising administering a pharmaceutical composition and/or medicament of the present iaventif . In one embodiment, a method of treating pain in a subject may be provided by aimmiste*¾g-a p simesutteai eornnoslticn and/or medicament of the present invention.

A fan &i aspect of the present invention ½ directed to a method for purifvicg PAP feem a mixture. The method may mdud¾ : -i ' n one embodiment, purifying PAP Horn a mixture b subjecting hie mixture to two or more chromatography steps chosen from anion exchange chromatography, cation exchange chromatography, mixed-mode chromatography, and bydfophothe mteractiors : ehro aiogmpby ; thereby providing purified PAP,

Another aspect of me present k entkm is directed: to a method of enhuring a mammalian cell expressing a recombinant PAP. The method, in ot c embodiment, may inciude culturiJig the mammalian cell in a defined serum-free base medium during a growth phase and maintaining the mammalisirce! in the base medium dtaring a production phase by supplementing the base medium with a efined^ sermmfree feed medium.

The- foregoing and other aspects and embodiments of the present invention axe described in detail ¾felow< ief i>es crip fm' n af the 1); ¾y. hm

F gure I shows a purificatio method according in embodiments of the present indention.

The present- invention iE now be described mom fhlly hereinafter. This invention stji- ho wever, be em odied in different forms and should not be construed as limited to the embodiments set forth herein. t¾ ie¾ these embodiments are provided to tally coavey the scop of the i ndention to those skilled in the m.

he ternnnotogy used in he desciiphon of the invention herein is for the purpose of describing particular embodiments only and is not Intend d: to b limiting of the inveniion., As used ia the description of he invention and the appended -claims, the singular forms

-and "the" are intended to include the hs forrns as well unless the context deafly indicates otherwise.

Unless otherwise defined, all terms ; (inchaiing; technical and seiefitifde terms) used herein have the same meaning as -commonly understood by one of ordinary -skill in the art to which ihi¾ invention belongs;. M will be: further understood that terms,, such as those defined in commonly used dictionaries, should be interpreted as avi g -a meaning that is consistent with their -meaning in the context of tile pteseat application arsdi relevant art and should not be interpreted an. idealfeed or ανψϊν formal sense unless expressly so defined herein, The termko!ogy used in the description of the invention herein is for the pnxpose of deserjhing particular embodiments onl and is not intended to he limiti ng of the in vention.

Ail publications, patent applications, patents d other re ferenues cited herein are incorporated by reference hi their entirety for alt purposes relevant to the sentence and/or paragraph in which, the reference is presented. In ease of a conflict in terminology, the pmmxt specification is controlling.

As used herein, "and/or" refers to and encompasses any and all possible combiMiions of one or more of the associated Hated items, as well as the iaefc of combinatio s en interpreted in the alternative ("or"};

Unless the -context indicates otherwise, it is specifically teaded that he various fcatores of the: invention desenfced erein can be used in any oomhinatiato

Moreover, the present invention also contemplates thai 1ft some etnbodmients of the invention, .any feature or combination, of features set forth heceiB- can be excluded or omitted. To illustrate, if the specification states that a complex eourprises cnmpOneMS A, B and C s i is speeificaiiy intended that my of A, B o C . or a combination thereof can be omitted an disclaimed,.

As used herein,, the transitional phrase "consisting essentially of (and grammatical variants) b to. be Interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novo! ehaacieris-tic(s) ! ' of the claimed invention, 8 , In re H 537 M 549, 5Sl-5¾ 190 il.S.F.Q. 4M, 463 (QCf A 1976) (emphasis in the original)! see also PEP § 21-11 ,03. Thus,, the ternr "consisting essentially of as used herein should not be interpreted as cqraivafent to "e-ontprisiag."

The term 5i shout," a-S: used herein hen referring to a -measurable- value, suc -as an ¾mo¾ttt. or concentration and the like ¾ is mnt to refer to variations of up to - ± 20% of the specified value, snob as, bnf : nst l½ited to, i 10%, ± 5%, i ].%, ± Q.SfL oreveft i of the specified : value, as sll as the specified value. .For exa le, "about X " where X is the t»easux¾ble v#u<vean mdndc ' as et as -a variation <£±2Q% S *.. 10% > ±■$¾, ± 1%,: 0,5¾ s or Q, 1% of X. A range provided lie-rein for a measurable value ma incMe any other range and/or individual value therein.

Provided herein are methods for producing PAP and/ar purifying PAP {torn n sample or mixture. The methods of the present mve ioncmay be used for both small and large scale PAP manufacture, roduction and/or purification. n ..some -emba eais, the methods described herein provide for a large scale manuraeture.. production, and/or purification of PAP at he 100 L or ore bioreactor scale.

The production and/or purification methods prcm ecl herein ; ruay be used to produce and/or purify PAP fro any organism that expresses PAP, ¾ certain embodiments, PAP may be obtained. from a mammal, such as. but not limited to, a ' human . , bovine, rat, and/or mouse. P AP nniy be a uatur&liy occaming PAP. A "imt raliy occurrin PAP" as used he ein refers to a PAP that may be isolated f si the orgaaism from which ¾ is derived, such, as, but pat limited to, a m mmal, .Alternatively*. PAP may be obtained b expressing it in a " host cell, such as but not limited to, a mairirnaKan 5: yeast,, bacteria,, or Insect, host ceil, and thus may be a recombinant PAR. Recombinant PAP may be expressed by cells in culture with the culture having PAP titers (i.e. , a PAP c-oncernraikm) of at kast about 100 mg/L, 150 mg/L, 200 mg L 5 250 nig/L, : 300 g?L 350 mg/L, 400 mg/L, 550 mg , SOQ mg' or more. Imoerta embodiments, .recombinant PAP may be ex resse .in. ceil culture in a range of about 50 mg/L to about 700 mg/L or about 100 mg L to about 500 mg/L, The eooseutmion of PAP is a sariiple or eornpositiou may be determined using methods -known;- to those of skill in the art. such as, but -not. limited to, by using enzyme activity assays * In some embodiments^ PAP may be . a rccombiuant, human PAih optioualiY obtained from a mammalian cell. Recombinant PAP produced in a host cell may be round ay a soluble protein, an msolubie protein, or any combination, thereof ou-lmiitiug examples of human PAP that nra be used with the resent invention include, but are not limited to, those corresponding to Accession &'ø£. P1S109.3;, AAA60021. U ΑΑΒ60ίί40, AAA69694.1,. AAAb0022, L XP . 005247624, 1, Ν _00¾09 ; 4, P i 5309 , 1530½fP 15309-3, SEQ IB NOri , and SEQ ID O:2 r

The production and/or purification methods provided herein may b used to produce andAa pariiy secretory and/or transmembrane PAP that is optionally soluble. "Soluble'.' as used herein means that at least a portion of the PAP is; rio present insoluble protein aggregates. In some embodiments, a soluble PAP may be dissolved in a solution (e,g;, cell culture medium, bodily fluid, buffer, etc.) in which, it is present to provide a clear to slightly ppaleseertt selmion. with no visible particulates, as ssessed by vis¾pi inspection. In some embodiments, PAP may fee th secretor fariri ' of PAP, ¾ρί¼τι¾ soluble, fu cert in em diments, PAP may be hieoam. secretory PAP, optionally soluble, and tile secretory, optionally soluble, human PAP may be recombinant.

"Purif inn" as Used herein refers to removing, isolating, separating, atid/or the like PAP in a s pl? or mixtu e fern at least one impurity or eofti&abuant present in the sample or mixture. The terms "sample" and "mixture:" 1 are used interchangeabl herein and: may Include a eoniposition eomprising PAP. il irfjp«rities ,i and %oniai nams'', arid their grammatical iarhs, are used interchangeably herein and include, but are not knitted i cellular components (e,g " host ceil components, · such as proteins, : nucleic acids, and/or lipids), bodily tissues or. fluids, and ox culture medium, PAP ma have a purity of about 50% or more, such as, bat. wA limited to, about 55%, 60%,. b5%, 70%, 75%, 80%, 85%, 90%, % %, 92%, 93%d94%, 95%, 9§¼ > 97% } M% 5 99%, ox more. In some embodiments, PAP may have a parity of greater than 95%, 9-g%, 98,5%, 99%, or 99-5%. nertam em dimeffis, PAP may have a purity of about ό¾¾ to about 100%, about 75% to about 100%, abord 90% to about about 95% to about 100%, or about % to abou 100%, PAP ma be purified according to a metho of the present invention,

A. method of the present Invention may provide a substantially purified PAP, "Suhstaritiaity purified" or ubsianriaUy pir 'b as used her en¾ rereis : :to a. PAP that has a purity of at least 50%. ¾ some enibodiments, a substantially punfied PAP ' inlay have a purity of at ' feast 60%; in certain embodiments, at least 95%; and in yet i rther embodiments, at least 98%, Thus, sebstauiia!iy pare PAP may be at least 50 free of Impurities,: Spelt as, but .not limited to, host eel! proteins .

A method, of the present veniiomrna p-ovide oompospdon compriairig PAP that has a host cell protein (HCP) content and/or Host cell PNA (HCB) conten : of less than about: 300 ng mg PAP S such as, hut not limited to, less than about 250 ngteg iPAP, 200 ng mg PAP < ISO ag/fng PAP, 11)0. ng/mg PAP, 75 ng/mg PAP, SO .ng/mg PAP,. 750 : pg/mg, 500 p /rng, 250 pgbmg, 100 pgdiig, 50 P;g/mg, : or 25 pg/mg; In som embo iments, PAP ma have host eeli protein content of less th n about 100 ag host- cell protein per mg PAP arid/or may have a host cell DNA content of !sss-t a about 180 pg !iost cell IMA per mg PAP. In some embodiments, a method of the present invention may pro vide a composition eonir sing PAP that has la host ceil protein content and/or host cell DN content of less d an about 100 ppm or 100 ng/mg. PAP; in certain embodiments, less: than about 50 pp or 50 ng/mg PAP in further embodiments; less thas abopt ' 25 ppm or 2S Bg/f»g PAP; and in yet f rther embodiments, less than a out 10 ppm or 10 n mg PAP.

The overall or total reco-vety fie., yield) of PAP after a metho of the - resen ¾ve ioa and/or after one or more step(s) in « method of the resent invention ma be about 20% or more, such as, but not i¾«ied t . about 25%, 30%, 35% y 40¾, 45%, 50%, 55%, 60%, 65%, 70%, S %, 85%, ?8 i. 93 ¾■ moret in some embodiments, a method of the present nvention .may provide an overall recovery otPAP of about 20% to abotrt 95 , a ou 30% to about 90 , ..about 3§¾ to about 75%, or about 30% io 50%,

T&e Kmoval of impantfes may result i¾ enrichment of PAP. "Enrichment," "emiehkg," arid grammatical variants thereof as used herei refer to an increase in the percent of PAP m a sanapfe Accordingly,, er iehiaerit of PAP ooeum when the percent of PAP is increased in a sam le after some manipulation of the sample, such as, lot example, subjecting he sample to one or more chromatographic steps. In some embodiments, a sample comprising PAP may be enriched by at least about a .IQ-foldtfcdttetf n. of Impunties, such :¾s » bat not limited to, bes cell: ¾tein¾ In he sample. ¾ certain enifeadimeiits, a saai le comprising PAP may be enric ed by at least about a 20-doid, 3ϋ-·ίο¾ 40-ioid, S9 b¾/S0- toli, 70-fold, gO-fold, . ¾4M t I OO-feid, or more reduction of impwities m the sa ple.

Methods: iiaowfi to ffiose of skill ia the an may he msed to determine the purity of P AP and ot me d reduction: of iffiperities m a sample comprismg PAP:, for example, PAP ma b analyse by gel electrophoresis, such as, but not limited: to, SDS- AOE, to detemwne the .purity of PAP, Assays and -methods:, such as, but not limited to, those described in US< Patent Application Publication Mo, the contes s of te!i are litcorporated herein by reference in their entirety, ma be used to measure imparities, sueh as, but not limited to, host ceil proteins, in a sample comprising PAP fld to calculate the fold, redactio of impurities. certain embodiments^ host eel! proteins may be detected, for example, b immtinoche ical methods iismg polyclonal antisera,

According to some embodiments of the present Invention, Methods of purifying PAP irom a: mixture eomprisipg PAP are provided, he mbitme may comprise eell culture medi and r a buffer. method of the present invention: may incl de one of more piniilearion and/or chromatography steps and/or techniques sueh as, but not limited to, til tiOB; :e ti fu ati 5 precipitation, phase separation, affinity p¾tifieation, gel. filtratiom ion exchange ebroma ography (fi,g,, anion and/or cation, exchange cl omatography). bydtophohic mterastioo ehrftrnatography, high performance Hq id chtramoiogrs h (flPLC), m xed-mods cliromotograpby, and amy combination thereof; ¾ some embodiments, a-

aaiofl exchange resm s a cation exchange re¾ a mixed-mode resbp. a¾d¾» a hydrophobic imsx tlm resin:, ,! Coata£tmg !f m tssed herein in regard to a

•placiiigj. adding, loading, ;mkifig, washing, ehrom ograpiiicaily eontaPimg Pod the like the mixture with a chrom togr phy resm, -Cteomatographicaii contacting'' as : used herein refers to contacting a m xture to be separated with a ehforaaio raphic raedm -, such as, hut not limited to, a res n, osing my mode of chromatography described herein and/or known the an. Modes include, but pre mi limited to, batch-inode and colunui cltromatography. Appropriate column dimensions cm be deiembsed .those s Iied in the art. Chromap^grapbically eontacfmg may be fefl¾et¾d ^o ft ^and^r iacu¼ti«g the: mixture pip in, and/or within the medmm : * li erhig the mixture thtotiglt f e medium; or by any other naeans known to those- of skill in tire art.

method of the present invention may comprise two or more chromatography steps, such; as, but net limited to, two, t ree:, feat, five, sbp seven, or more chixmiafcgraphy steps, In some embodiments, a taeihod of purifying PAP m comprise providing & mijdnre comprisin PA¾ and puriiyifig the mixture by safajecting fee mixture to two or more shroffiatogrpphy steps chosen irom or selected from: the gron consisting oP amoir exchange clrrctmatogfap ly, cation exehange nbromato taplty, mixed-mode chromatography, and hydrophobic interaction ehfomatography to rovide a purified P AP, As those skilled in the art will understand;, the two of more chroinarogxaphy steps may be carried out in any order,

In some embodiments:, a BAP purification method of the present invemioti comprises at least four chromatography steps,. which may comprise the Mo ing chromatography rnedium: anion exchange chtomatogrirphy,, cation exchange emxnnatography, mlxed-mede chromatography, and bydrophuhie biteractioa eliramatography, in certain embodiments, each of the tMswiug c romatogra hy media are used in the following order: anion exchange chromatography, catio o change chromatography, mixed-mode cbromatogfitphy, and hydrophobia interaction ohromatography., but other steps, iapinditig othe chromatography stsps, ma be included betwesnonc or more of the chromatogra hy : steps. A buffer may be sed m a method of the. present invention, in some emb di enSj

eluate may com rise at least a portion or all of me FAF e luted after the anion exchange ehromstography step,

A method of the present invention may comprise contacting a mixture comprising PAP with a -first chromatography medium; Mi&g at least a ort on, of the PAP froin the first chromatography medito. to obtaih a firs t PAP sample; contacting at least a portion of the first PAP .sam le with a secon chromatography medium; and e umg at feast a portion of t e: PAP from the second chxo atography med um to obtain a second PAP sample. The chromatography steps may he repeated with, the same and /or dif¾irfc#t chromatography media uniifPAP with a desired; purity is obtained.

la same embodiments, method of th p esent venfem comprises competing a rmxt jre CGtaprisiftg PAP with an anion exchange chromatography medium; elating at least a orti n: of the PAP fr¾m. the anion exchange chromatography mediam. to obtain a .fat PAP sample; contacting at least a portion of the Erst PAP sampl with a cation exchange cfn smatography medium; elnting at least a portion of the PAP from, the cation exchange chroffiatogmphy medium t o obtain a second PAP sample; contacting at least , a portion of the second. PAP sample with a mixed-mode chromatography medium; elating: si least a porhon of the ' PAP from the .mixed-mode chromatography medium to obtain a third PAP sample; contacting at least a portion of the third PAP sample with hydrophobic in eraction •chromatography medium; and elating PAP from the hydrophobic interaction chromatography medium to obtain a fourth PAP sample,

E¾empiary material that .may be used in a purification e o of the present nvention, such as, hot not limited to, chromatography medium, are described !ierein.

Anion exchange chromatograph relies on chaige-charge mteractions bevween the components in the s mple s , such, as, .for example, proteins, and the charges immobilized on the. anionic chromatography i&edipma In anion exc ange chroma ography, the binding ions of the eonrpooenis are negative and the immobilized, fhnstional group of the chromatography medium Is positive. Exemplary anionic exchange chromatography medium iudude/but are not limited to, resins,, mouoiithsa or membranes:. In seme embodiments., n anionic exchange chromatography roediimi is in the ferm of a column comprising anion exchange chromatography resin,

Strong anion exchange media may comprise a, quaternary ammonium ion ami weak anion exchange media ma comprise a tertiary and/or secondary amine, functional group, sueh. as a diethylaminoethy! (DEAE) group:. Exemplary ttmcuoraai groups anion exchange medium ma comp ise include, hut are no fh ted ¾¾ mmt¾a¾ty arimieaiitmi groups- sun as qmifere¾y ¾3k »iaes- mA quaternary kyi k ol amines, dieifeylamdfie grou ; diet!iykmiiiQ fQpyl groups, i mo groιφs :teimeίhylammόnirø.e¾Ily! reups,. trimei ylfaeiizy! affiisomunj groups, diinefeyietlisiiolhetey! ammonium- gou s, aisd polyamine groups. Amoiuexeharsge s may com ris a eopss-rion, such as, but i limited to, a ehloride ios (C1 " I -which may maintain efeeiron w iy.

Exeinplary anion exehauge -membranes- ½el¾i¾ b ar&- apt limited to. SARTQBlAiD® Q .from Sarterras, MUS¾J CJ® Q from- Pail Technologies and INTERCE T® Q iBem!3«¾ s item Mt ipo :,

Exem lar a e exchange resins, ¾e!«de,: : hut are not liput k>„ those that comprise quaternary armiaoethy] (QAE) ffioieiy such as, ¾>r example j: TOYQPEA LS ) QAE

. : 0-· Ati anion .exchange chfdi»i¾iography step may be carried out using a buffer, suc as, bat not limited .to, one or more of he buffers described herein, ' & some embodiments, a ris bufifer may be used k ¾¼ieii .n with as anion exchange ck'omatGgtaphy ste o:f a method of the present invention. %as s a. mixture comprising PAP may eoniprisa a Tiis buffer, T¾6 Tvis buffer ma comprise Tm-HCi in a concentration from about 1 ni to about 1 M Trix- BCl, such as, but not limited to, a concentration of abonl 5 w , 10 na , 15 mM, 20 aM, 25 siM, 30 HiM, 35 mM, etc. Ik tris buffer may ha a pH of ahom 7 to about ¾ such as, bu sot limite to, a ρίΐ of about 7, *!.S, 8, 8,5, or & la som embodiments, a mixture comprising PAP in a Tns buffer having a Uris-HCi coneeniraticm of about 10 raM to about 35 mM and a. pii from about 7 to about S may he contacted with an anionic exchange chromatography resin.

Elubon from an anion exchange resin may be achieved: b increas ng the conductivity of the mobile p¼ss f such, as, by adding a salt ,, sodium chloride) to a baf&t to form an anionic elation buffer. A Method of the present invention ma comprise contacting an aaiortic exofemge resin with two or more anionia elation buffers that may b¾ (he same and/or different. in some embodiments, .. PAP is elated ixom an anionic exchange by eantaeting the anionic sxcliange resim h an . an onic elation buffet,: The anionic eltstion buffer may- comprise a salt, suck as, but not limbed to, sodiani. chiorido, in: a eoncenaaiioa from about $ mM to about 2 M, about SO mM to about I M, about 50 mM to abant 100 mM, about 750 aM to about 1.25 , or about. iOtj mM to about 300 mM. I» -some embodiments, the anionic eiuiion biiffer comprises a birffer, such as, feat Pot limited to, a Tris baffer, and a salt

Cation exchange chroEiatogr&puy also relies pa charge-charge interactions between the components m the sample, saeh as, for example, proteins, and the charges immobilized on $se- araonic- e¾<ro^#i grap.hy . . m$3i¾m. in cation exchange ehasrhatograph , the binding ions of the eomponems a ositive;, and the InmmMliaed functional gro up of the eiaomafe graph y medium is negative. Exemplary cation exchange chromatography medium include, tat are aot limited to, resins, monoliths, or membranes, in. some embo iments, a. cation exchange ohroatatography me ium Is in. the form of a column comprising cation, exchange, chromatography fssim

Strong cation exchange media ma comprise a sulfonic acid group, such as sulxb ropy] (SP) . group, and weak cation exchange media may C mp ise δ weak ae d ^ such as earboxymethyl fCM). Strong cation exchange groans may have a wider pB ange■■compared to eak cation excharigers, Further exemplary fnnciianal groups cation exchange cinematography media may comprise include, bat are not limited to, arboxylie aeid

1* timet! onal groups and sulfonic- add limctiotiai groups; examples of such groups include, hat are not .limited to, sulfonate,. carboxyi.% carhoxymeihyl suifoiue acid,, suif iso uiyl, sulioeihyl, earboxyf suiphopropyi, sul honyl, snlphoxyethyl., and orthophosplaate.

Exemplary eabon exchange chiOmatogra hy media include, but are- mi limited to, those having a sulfonate based group (e.g., MONO S®, MlmS, Source™ T5S and 30S ¾ SP 5FJ>BAROSf¾ Fast Flow, aud SP SEPFIAROSB® High Peribmianee available from OB Healthcare, G^man ; ΪΟΥΟΡΕΑΕΕ® SP-&50S .and SP-656M available irooi Toso!i Bioscience, Oermany; MAC¾ FREF¾ ) High 5 av il ble -few BioRad, California, USA Ceramic HYPER. D© S, TRiSACRYL® M and LS SF and SPHEKODEX® LS- SP available from Pall Corporation, New York, USA; a sulioethyi based group (Ο,Ε,, MC CfOGEL® SB from EMD ΜιΙΙίροίο, POROS® (S-IO aiid: &-2G available ffota. Applied Biosysiems); a siipbopropyi based group- l ,g, } TSK-GEL® SP 5PW and SF-5PW-HR. available from t o POROa® HS-20 and available irom A¾>f Biosystem ); a sulfoisobutyl based group. (o . y FRACTGOEl® EMD S03 available fern EMD and ESHMUNO© CPX available iforri EMD Mi!lipoi¾ Germany);: a s lfbxyethyl based group (e,g, 3 SES2, SES3 arid ' EXPRESS-ΙΟΝΦ S available -from Whatman), a. carboxysnslhy based group (e.g., CM SEP:HAR.OSE# Past Flow available from GE Healthcare, HYDROCELE® CM available from Bioefenu Labs toe;, MAGRO-d¾EP® CM BioRad, Ceramic. HYPSR ΏΦ C , TRlSACRYt® M CM, TRISACRYL® LS CM, from Pali Technologies, MATREX® .Ceiluffoe C500 and C200 from Mi!ipore, CM52, CM3Z, CM23 and EXPRESS-ION® C Item Whatinau, lOYOPEARL® CM-650S, CM*650M sod G -650C from Tosoa); sulfonic aird caxboxyMe acid based groups (e.-g. BAKERBO D® Carboxy-Suifon from AT, Baker); a carboxyile acid based group (e.g., WP CBX from J f T Baker. DOWEX® MAC-3 from Do li reid Separations, AxnbeBHe™ Weak Caiior* Exchangers,. OOWEX® Weak Catiaa Exchanger, d DIATOM® Weak Cation Exohangers frdra Slgma-Aidrfch ψφ ΈΚΜΤΐύ&ΒΐΜ EMD COO" frsm BMD i &te}; a sulfonic acid based group (e. g, : , Hydrooell SP frfttp Bioobrom Labs loo, DOWEX® Fine Mesh Strong Acid Cabon Matrix from Dow Liquid Separations, . UNO SPHERE® S, WP Sr ibmo fwm I X, Baker, SARTOB D® S membrane from Sartorius, AMSBRBITE® Strong Cation Exchangers, DO EX© Strong Cation and I)!AlO © : Strong Cation Exchanger from Sigina-Aldric% and a orthophosphate based groap (e.g,, PI 1 from Whatman).

Exempkty cation exchange membranes include, but re not !uni ed ¾¾ SARTOBIMD® S (Sartorius; Idge o d, NY),

A2- Further exernplaty eMk & exchange chromatograph media acco.r4¾. to embodimerits of the present invention mcigde, bu are -not " Kmked to, those described ½ InleBiat!oaal P blkatfou . WO: 2011/1 0110 and U,S. Patent Application Publication No. 2013/03171 7 » he contents? of each of which are incorporated herein by reference: i inch eftticety... in some erubo imente, a cationic exchange chromatography medi m may comprise a hydrophi!ic polymer matrix, siich as, bat not limited ¾¾ a poiyv ylether. polymer. The resin .may comprise a tentacle st uc ure comprising the hydropbilic polymer matrix with, cationic exchan e rursctional gte¾ps, sucls as, but not Krohed. to, milibisotetyi ilffictional groups, at the e»d of the tentacle structure, in setae einfeftdime s, a cationio exchange resin in embodurieuts of the present invention rosy comprise a sulfoisobuty! functional group, A eauonie exchange chromatography -medium according to embodiments of the present ibventloii ma comprise a strong canonic exchange- resin.

A cation exchange chromatography ste may be carried out using a buffer, such as, but set limited to, sae or more of the . buffers described herein. In same em odiments, an. acetate buffer, s, but t limited to a . -sodiu aceta e buffet, may be used in coruunction: with a ' cation, exchange chromatography step of a method of the resen invention, Thus, mixture comprising 3¾P may comprise an acetate buffer * The acetate buffer may comprise sodium acetate (MaOAo) in a eoneenniatiori of about: I tfiM to about 1 NaOAe, such as. bed riot limited to, a coneemratiou of about 10 mM, 25 mM, SO mM, 75 mM, etc, The acetate buffer may have a pH of aboiat 3 to -about 6, such as, hot not iiadted to, a pH of about 3, 3.5, 4.5, , 1,5, r 6. in some e bodime ts, a mixture comprising Έ in an- acetate ' b¾t& having i aOAc ooricentratioo: of about 25 mM o about 75 mM and a pH &om about 4 to aboatS may fee cantaeted with s catioaic exchange chromatography resin,

Htttior!: irons a cadorsic exchange resin may be achieved by iaereaaiog the conductivity of the mobile phase, such as, by adding a s it -^ ., sodium chloride) to: a buffer id form catioftic eiutiou buffer. A method of the present invention may comprise contacting a eati nio exchange resin with two or more cation el don &uffe*s thai may be the same and/or different m some embodi ents;, PAP is drited from ¾ catio e exchange resin by contacting the caiionie exchange resin with a eationic elation, buffer. The cationk ehitiou buffer may comprise a salt, such as*. -but not limited to, sodium chloride, in a conceormtiorj. from about 5 mM 2 H abdtjt 50 mM about IM, about 50 mM to about 200 m , about 200 ¾M to about 600 M, or about 200 mM to about 400 mM. In some embodiments, the cationic elation buffer comprises a hufler, such as, but not limited to, as acetate buffer, .and a salt. M d-t&ode cinematography medium ms be used in. -efeomaiography step of the present Saveii^m Mixedmiode efrromaio raphy medium comprise a support or solid

exchange, hydroxyapaiite, af njty, size exclusion, and/ot hydrophobic interactions.

Exemplary i ed^n ' ode ehroinatography resins include, but are not limite to,. BAKERBO i ) ® ABx™ (J. T. Baker; ¾§llipsb»¾ Kl% ceramic hydf&xyapatite (CKT) type I and II resins and fluoride hydroxyapatite resfes (Bio&ad; Hercules,. Calif.}; MEP il erCeI ¾i Md ΜΒί HypferCdt™ (Pall Coiporation; East B¾ N.Y.); Gapm™ M C > Capto™ Adhere, HEA H erCeJW PPA. HvperCel™ GHT™ ceramic hydroxyapadte, Idavia™ ePnme^; aad Bio-Gel HT, Bie-Gei HTP, Biorad, Hercules, CA, urther exempla y -m!xed- ode chnQmatography rnedk mcltide, -fe¾t are not limited t6 > those described: in U.S. Patent No. 7,999,085; U.S. Paten Applioadon Fablicatiea Nos. :201d/Q0817Q0 arid. 2013/8317197; and iotemaiionai Publication No. G 201 ! /iSQl l O, the contents of each, of which: are incorporated lie- n by reference m thei entirety,

According to some embodiments of the? present invention, a m x -mo e diro atography medium may comprise g ceramic hydroxy apatite (CH or ceramic O-uorapstite CFT) resin of Type I or Type II. The CHT st -CPT resin ma comprise one or mom ionic gronps chosen fr m or selec ed' from d e group.- consisting of Ca' ''* f-Q^ OH\ and any eo nation. thereof. The resin may have an avera e particle/ iameter of about 1 % 2(1 40, or 80 m crons. The choice of hydroxyapatite or :fiuorapaCite s the type, and average particle diameter can be determined by the skilled artisan. In some embodiments, a chromatography step of the present Invention .comprises a CHT mixed-mode resin, In certain embodiments, the CHT m xed-mode resin s Type IL

A. mixed-mode chromatography ste may be carried out usin a- buffer, such as, but not limited to, one o mote of the butlers described herein, la some embodiments, a EfEPBS :b¾fer may be used in conjunction with' a mixed-mode eliromatography step of a method of fee p esent invention. Thus, a mixtttre comprising PA may comprise a HEPES bnifer. The HEPES buffer may bave a concentration from about I mM to about 1 M HEPES.. sach as. but not limited to, a concentration of ab u 5 mM, 10 m , IS ¾¾ 20 mM, 25 mM, 30 mM, 35 mM, etc. The HEPES : buffer may have a. pH of about 6 to about: 9, such as, hot not limited to, a pH of about , f S, 7, AS, $, « or 9, Imsonae embcicii en s, a mixture comprising PAP In a HEPES .buffer having a concentration: of about 15 mM to about 35 mM HEPES and a H from about 6,5 to about 7,5 may be contacted with a m xed-mode chromatography resim The ¾EFES buffet may comprise a. salt optionally in. a commtm m of .about 0,5 mM to about 50 mM or about 1 m to about 20 mM,

Bl ior. from a mixed-mode · chromatography resin may be achieved by methods .know to- those of skill ' itt. the art. A method of the prssenl invention may comprise contacting a mixed-mode clmomatography resin wife a mixed-mode elntion buffer. In . aome fembodiments PAP may be eluted from, a mixed-mode chromatography resi.fi by- Contacting the mixed-mode chromatography resin with a mixed-mode chromatography eiuti m buffer, " The xe -mode eiution uffer- may comprise a salt, such as, but not limited to, sodium phosphate, in. a concentration from about 0, 1 mM to about i . about 1 mM to about lO nn h or about I mM to about , 500 mM. in some embodime ts, the mixed-mode eiution buffer comprises a . ' buffer,, such as, but ot limited to, so pigPES buffer aid sal such a% hut .not limited to, sodium phosphate.

Hydrophobic interaction chromatography may be utilized according to some embodiments of the present im entiom Hydrophobia interaction chromatography media may comprise a stmpost ifii a hydrophobic ligand. The support may comprise a base matrix (e.g., eross-lirjjced agarose or SYfithebe copolymer material) to which hydrophobic Kgands (e.g., a!kyi or aryl groups) are coupled. "Alky!" as used herein refers to C s .20 inclusive, linear ie./ "shaiglb-ehaiif'}, branched, or cyclic, saturated or at feast -partially ¾nd- In some cases folly un¾aiurated (i.e. s alkenyi and aikyny!) hydrocarbon cha ns, f ' AryF as used herein refers: to an aromatic subsbiuent that may be a single -aromatic ring,, or multiple aromatic rbigs that are fused, together, liuked covale ly, or linked to a eornmen group, such as, but not limited to, a methylene, o ethylene moiety, T& term "aryl" specifically encompasses heterocyclic- aromatic compounds and .substituted aryl,c0mpound¾ s such as, but nat bmited to aralkyi, The hydrophobia -Ugand may he available to interact with hydrophobic components,, such ¾ , for example,, hydrophobic amino acids of a protein, Exemplary hydrophobic figands or functional groups include., but. are not limited to, butyl -ether, propyl ether, phenyl phenyl ethet. and any combination thereof

Exemplary hydrophobic interaction chromatography resins include, bat are not limited te Phenyl SBPHAROSE® S fast Flow; Phenyl SEPH AROSE® High Performance; Phenyl SEFHA .OSE® High Sub; Oetyl SEPHARDSE® High Performance (Pharmacia. LKB Biotechnology, A¾. Sweden); PRACTOGEL® BMP ' Propyl or PRACIOGELi) HMD Phenyl (EL Merck. Germany); MACIiO-PREP® Methyl or MACRO-PREP® t- utyl S ppofts 0!o~R d, CA); WP Hi-Prspyi™ (Cf) (if T, Baker, NJf ; and TOYOPEAilL® ether, henyl or butyl (TosoHaas, Pa,),

A rnethod of he present invention ansy comprise Cositaetki -a- ½ ·ώ»ρ¾ο ' « interaction. chrop^Stogmpiay resin comprising eross-Hnked agarose with a mixture comprising E4B. In some embodiments, the Hydrophobic Interaction chromatography resla may com rise an axyl functio al gronp, sueb as . , Hat not limited to, a phenyl ' functional group,

A. hydrophobic interaction ...chromatography step: may b carried out vjsing : a taifife*, sueh , but. ¾ t limited o, orie or more of the buffers described herein, Irs. .some embodiments, a phosphate buffer may be used in conjunction with- a hydrophobie interaction ohroniaiography step of a method of he: present Invention. The- phosphate buffer may be a sodium phosphate monobasic (Na¾PQ-4h sodium phosphate dibasic :( ¾HP04} > potassium phosphate moriobasic ( H^PO,)}, or a potassium phosphate dibasic (SL2MPO4) buffer. Thus, a mixture comprising PAP may compris a phosphate buffer. In some embodiments . , the phosphate buffer -comprises. ¾ίϊΡ0 . The phosphate buffer may have a corjeentfation Sam sbo . 1 m to about 3 M K^HPO*, such -as, bat net limited to, a concentration of about I to about 3 M, about .1.5 M to about 2.5 M, about 7-50 mM to about 1.5 M, about 1 .mM to aboei 308 rhlvL aboat 400: mM to about 700 mM. or about tOQ mM to about 1 Μ ' Κ 2 Βί > ί¼. The phosphate buffer .may have a pH of about 5 to about 8.5, such as,, but not limited to, a pH of hosi Si S.5, 6, 6.5, 7.5, 8, or S3, in. some crnbodirnents. a mixture comprising PAP in a ¾HP0 4 bufibr having a concentration of about 750 juM to about 2.5 K 2 HPO 4 and a pH. ftotn about 7 £0 about 8 may be contacted with a. hydrophobic (ntcractior chromatograpby resin.

Methods for elutihg a protein from a hydrophobic mteraction chromatography resin are known to those of skill fo the art. Hydrophobic mte chons imy be e&hauced or stren hened by buffers: with high ionie strength, Therefore * protein, such as PAP ma be duted from a hydrophobic interaction chromatography resin by reducin the strength of the hydrophobic interaction;, such as, hut not limited to, by reducing f¾e- ionic strengt of the mobile phase. In some emb diment^ PAP may be eluted from a hy of ophobte interaeb on chromatography resin hy contacting the hydrophobie interaction ohromatography resirt with a hydrophobic interaction chromatography elurion buffer, The ' hydrophobic, interaction efu on buffer uay have a lower ionic strength than the ionic strength of the mixture comprisin PAP that contacted the hydrophobic interaction; chromatogr phy resin, hi some embodfxnenis s the hydrophobic-. interactiori¾ eiution buffer comprises a phosphate buffer, such as one or more of those described herein. -In certain embodiments, the hydrophobic interaction emtion buffer comprises Κ ΗΡΟ^ The ¾HP ¾ buffer may have a concentration from about SO ivM to about 500 mM or about; 100 rn.M to about 300 mJvt

A M purification method of die resent■ invention may comprise a filtration step, m& as, but not limited to, a microil!tratlon, t^irafiitiratian/djaiiit!-ation, angential flow filtration, and/or alternating tangential How filtration step. In some embodiments, a method of tire present- invention comp ises am ultt¾liItrgiion diafiltratiori ste . The iUtmfi¾ratiori/di¾ii!tratiori ste may ' be carried out as a eontlimona diaf iraticin step, a diseoxninuous diafiitration stop by volume reduction, and/or a diacomimjous diaBltraiiou step b sequential; dilution. An. vtirrafiitraiiors/diaillinjtioi step may be used wo o mare times, such as, but mi I ited to s three, four, five, or mors times, daring a method of the presen invention. An lti¾fiitr£ ion/iiiafiltrat;or; step may bo used to concentrate a sainple, to c mg$ the composition of a sample (e.g., to buffer exchange a sample), and/or to remove a oomponent (e.g., a salt) or an impurity in sample. Irs some embodiments, m ltiafiitratiori iaftlttatioa stop may be use to ohau e the composition of -a sample comprising PAP, The sam e may be modified to have a difieiint buffer aud or pit. The sample comprising PAP may be modified te comprise a saline solution, such as but net limited to a : phosphate buffered saline solution and/or a .it.9% saline solution. The satin© soMion may tee a pH of about 4 to shorn & * siich.as, but net limited to, a pfi of about 4,5 to about 7 or about 5.

Exemplary eltrafi!tra ioa/dkfiitration material include, hut are not limited to, membranes and filters such as membranes andror filters comprising cellulose such as regenerated eelMose, polyethersuifene ch s: modified poiyeih&rsulfoue, and/or hydrophilic polyvii-tylidene finonde. Exemplary commercial tiltrat ltraiion/diafiltraiion materials include, ut are not limited io,. BELIICON® 2 microfntratlou cassettes .with &lO AX¾ UOTA.CEL® PLC, and/or 0URAPORE membranes available from EMS) Miliipor e arid Allegro™ products from Pall Corporation, The: u!trafi tration/diafiltration membrane and/or filter may have a molecular weight cut-off, sne as, but not limited to, 10 kDa, 20 kl>a, 30 kDa, 40 kDa, 50 k 60 ¾3¼ 70 kDa, 80 fcDa, 90 KDa, 100 kDa, 110 H>¾ 120 kD¾ etc.

A method of the present invention may comprise a viral inactivatien step. A viral inaetivation step may be carried eut as a single step is a method of the present irsveotion or may be carried out mul iple times in a method of the present, invention, For example, a viral, inaetivation. step may be used two Or more times, such as, but not limited to. three, four, five, or more times, during a method of the present :ipveution. Any -s¾i table -vital im¾tiv¾tIon method may be used in a method of the present invention. .Exemplary viral mactivation methods include, but are not limited to, hear mactiystion, irramation sue¾ as gamma or UV irradiation, chemical treatment such as. viQi ' aa organic: Solvent, TweetH&O* Tri oB-X and/or lri(n- tyl) h©s0bate, filtration snob as nanofiltration, precipitation, and those described in international Publication Nos, WO 2008/03902! and WO 2008/098979, the contents of each of which are incorporated herein by referenced hi ihe entirely,

la some mhbdimerits, a .method of the present niveMion comprises a, viral mactmabor:. step that may be erformed or carried ovet wiib a d emlcah such as, but not limited to Tnton-X, in some enmo imenis^ a method of the present inventi n comprises a viral ina sivation step that ma be. performed or carried out with a virus removal filter. Exemplary virus removal filters include, bin are not limited to, PLANOVA® fibers available from ASM, Glenvie , IL and VIRESOLVE® available .from ίΠί α Bftierica, Mm. Tbe viral ia¾ctjvatian step may be pwfbrmed or carried ottt at any time doriag a metho of the present vention, In some embodiments, a viral inaciivabon step is performed or carried oat a ter tbe first du'orttatograpby step and/or after tbe last .chrom tography step. In certain embod ment^ a viral m&eiivabon ste is performed or carried oat after a mi n. exchange chromatography step..

A method of tbe present invention may comprise t o or more chromatogfapby steps, A chromatograph step of the presebt invention may be cam out under conditions suitable for at least a portion of PAP to be purified from the mixture. A chromatography step may be carried out at temperature of ahos 5°C to about 30°C. In some embodiments, a ciriomatograpby step ma be carried out at about room temperature, between about C to about 8°C S or between about 0 ¾ C to about 30°C. Sequential steps ia a PAP purification od/or. maaufactoiing method ' of the present invention may be continuous and/or discon mrioos.. In some embodiments, a method of the present invention, comprises a continuous purification method that optionally may uthtee the same flow rate, conductivity, •and/or pB for one or more steps.

Q or both of the bvo or more obromato raaphy steps may ©omptiae column chromatograpby steps. As those skilled in the art will recognize, a chromatography medium, .such as a column comprising earoma¾gfa by resin, may be ashed one or more times with an equilibration buffer and the :flow in-ougb fraction may be discarded The eomhbration buffer may be the saute as or similar to a buffer present in the sample to be loaded onto the eolmwi. The■ column dimensions may be determined by one of skill in the art. For example, as those sMi!ed n . the art will recognize, the amount of ebromafography resin to be used may depend, on the o veral l protein conten of die sample to be applied to the column. In some embodiment a chromatography step of a method of die present ention comprises a column having a. vol lane of about 5 ml, to about 1 L or more, stick as., but not limited to, about 10 m.L to about 50 m ' L about 40 mi to about 1,00 mL, about 200 mL to about 7$0 mL, about 500 mL to about: t p, or about 30 mL to about .500 »iL.

A coimnn chromatography ste of the resent invention -may comprise: contacting a sample with a chromatography resin resent in a column at any suitable protein load. Thus, the sample .may be loaded onto fee resin in the column, iu Som.e embodiments, the proteia load is ui least about 0,5 mg PAP/niL chromatography resin. For e¾am ¼. the protein load in may be about 1 , 2, 5, 30, , 20, 30, 40. S0 : 160, 150 or more mg ,PAP/mL chromatography resin, in certain embodiments, the protein load may be about 1 to about 20 mg PAP L chromatography 1 resin.

According to some embodiments of the present invention a mstliod of the present invention may comprise a method, for purifying PAP 100 such as described in Figure P. The PAP may he recoinbinar PAP. such as, but not limited to. recombinant human PAP. The recombinant PAP may be obtained from a cell culture, Thus, the method 166 may comprise the step of clarifying the culture of host cells expressing PAP 110. The arifieahon step 110 may comprise removing the hostneils, such as, but not limited to. mammalian host cells (e.g., human cells, Chinese hamster ovary ce!l¾ etc.), from the cell culture medium at the termination of the culture by depth filtration,

After the clarification step 110. an uitiafiltraiionAliaflltration ste 120 may be performed. The uitrafiltratiooAliaf l nation step 120 may be carried out t buffer exchange the clarified harvest. Optionally, the til rscfiitrati¾fl disiil¾¾t|QB . s ep. 120 may provide a buffer suitable for m anion exchange chromatography step 130. The clarification step 110 and/or tUs-' llxafilijatto^aifitoafi n step 120 may remove cells and ceil debris from the cell: culture.

An anion exchange chromatography step 130 may then be performed. Alternatively, a method of purifying P AP 100 may start with the anion exchange chromatography stop 130 ^Uhoui a prior clarification step 11 aud/or diraShrnh step 120. The ante exchange chromatography step 131) may comprise contactings sample comprising PAP, such as, but not limited to, tire clarified harvest from the clarification step 110 or the sample from the iihraiilKatlorPdjafiltratioEi step 120, with an anion exchange chromatography medium and at least portion of the PAP may be eluted from the aoton exchange chromatography medium. The anion exchange chromatography medium may comprise a strong anion exchange resin, such as, but not limited to, an Eshmuno Q or C) Sepharose chromatography resin. The yield or recovery of PAP in the sample after the anion excha ge chromatography

A 9- step I ' M mm be at least ab nt 50% s . such as, hoi not limited to, abotrt .6t% to a¾o¼i , Tie host cell protein: %vel in the- ' sample after the antec. e change ciiroBSatography step 130 may be reduced by at le st 30% v such , b . t ihxmsd to * by about 45% to about 62%>

Optionally, a viral hiactiyation step 135 may be performed or carr ed out with fife eluate from the amon exchange - chromatography step 130 (he,, the sample- ftomprisiisg PAP}, The viral ii activahon : step !3S may optionall comprise a. chemical viral maet ation. For example, a chemical vital uiaetivation. ma comprise contacting the gruate from the viral inaehxation s ep with 1% Triton X QQ ferahooi ί to about 2 hours at room temperaitwe.

The eiirats from the nion exchang chromatography step % or the elnate after hev iral inaehyahon ste 135 (be., the sample comprising PAP) may he. -contacted with a cation exchange cinem t graphy medium and at least a portion of the PAP may be ehrfe f m the eatfett exchange chromatography medium in a cation exchange chromatography step 140, The cation exchange -chromatography .mechaoi rnay comprise a strong cation exchange nssin, such as, btrt not limited to, ari: Eshnmno CP chromatography resin. The cation exchange cinematography step 140 may e a step gradient process.

In some mbodiments, most of the imparities are removed from the sample composing PAP after the cation exchange chromatography ste 140, For example, PAP in, the eluate from the cation eXehaage chromatography step (i.e., the sample comprising PAP) ma have a purity of at least about 5if%, such t but not limited to, a parity of ax least about 60%, 65%, 70%, 75¾, : 80%,. 85%, Wo, 9M or mom, The recover or yield &f PAP in t e: sample after the cation exchange chromatography ste 140 ma be at least about 50%. such s, bat no t limited to, a yield of PAP of at least about 60%, m % s 70%, ?S%y 8 %, 90%, ormx>re or about 65% to about 85%. The host eeE protein level in the sample after the cation exchange, chromatography siap, 1.40 may be reduced by at least 75 % ? such as, but not limited

The host ceil protein evel in the -sample after the u rafil -aiimi iafilfration step 14S ma ¾¾ reduced by at least 40%, . . such as, but not lira bed to, by about 53% to about 65%.

The eluate from i& e bon exchange 'Chro atogra hy st«p 140 or after ltmfiitraiion/diailliratsan step 145 iTe,, the sample oon rising PA ) may be contacted with a mixed-aimde chromatography " medium and at least a portion of fftft PAP may be ed from the mixed-mode; e!mmiatography medium in, the ffiised-m de chmrflatography ste ISO. The mked-mode chforaatography medium may comprise a ce amic hydfoxyapatfte. The yield of PAP in the sample after the mixed-mode chfoinatogra h step IS may be at least about 80%, such as, but not limited to, about.95% to about 99%. The host cell, protein level in the sample atkt the mixed-mode chromatography step HO may be widuced by ¾ ieast73¾ i s ch as, but not felted, to, by aboai % to about 90%, PAP in the eiuate front the mixed-mode elm>m atography step 351) may have a purity of ¾t least abo t 10%, such as, but not limited to, a purity of at least about 85%, 90%, 95%, 96%,:97¾ s 9:8.%,. 98.5%, 99%, 99.5%,: or mm-

The ehmte from the mixed-mode ebaomatography ste ISO " (ie., the sample comprising. PAP} may then be contacted with a hydropho ic:, interaction chromatography medium and at least a portico o the PAP may be ebfted bom the hydrophobic interaction chromatograph i a hydrophobic interaefiou chromatography ste 8. The hydrophobic interaction chromatography step 160 may comprise a step gradient process. The hydrophobic interaction chromatography mediu may comprise an aryl functional group* sueh as. hut apt limited to, phenyl. The yield of PAP in the sample after the hydrophobic interaction chromatography step .160 may be at least about 60%, such as, bra not limited to, about 78% to about 87%. The host ceil, protein level in the sample atet die hydrophobic interaction ahromatogxapby step 161) may be reduced by at least 80%, such as, bet not limited to, by about 90% to about 99%,

A ^irai inactivaticn step 3.65 m y be performed or carried out with, the oiua¾ from, the hydrophobic interaction chromatography step 160 (be,, the sampis comprising PAP). The viral inaebvatiori ste !&S may optionally cc-rapnse a filtration viral maetlvafiom For example, a filtration viral mactivatien may comprise filtering, such as, but not limited to, nanofi ering, the eluate from- the hydrophobic Interaeiiou chromatography step ISO. The yield of PAP in the sample after the viral mactlvabon ¾tep 165 may be at least ¾£eui 90%, . such a¾ but not limited to, about 9b% to about 98%. The host ceil proteiu level in the sample after the viral mae ivaiio ste 165 may be educed by at least 80%, such as, but not limited to, by about 90% to about 99%. An ul rafu&¾tidn dlafiitraii0n atep 170 may ¼ perfomed or. carried eat with the elaate &o& the vital mactivatiori step ½5 (i.e., the sample comprising PAP), The itfgiiitratioiv'diaiiltratjon: step 170 may be carried out to buffer exchange the sample

protein concentration of less Chan about 100 ppa¾ and/or a host cell riTN A conceMration of less than about 100 pg host eel *DNA per mg PAP. In some embodiments, PAP after the metboi 106 has a specific activity o about 200 to about 500 uniis/mg PAP, Thps, m some embodiments, after the .method 1 : of purifying PAP 100, the sample comprising PAP has a PAP purity of at least about 98% t a boat cell protein, concentration of less than about 100 pprm a host eell: rONA. concentration of less than about 100 pg host ceil riJNA per mg PAP, and/or a specific activity of about 200 to about 500 o itsAB PAP,

According to some embodiments of the present invention, a purified PAP m be ebtainsd word ng to purl fieaiioo method of the present invephmn The purified PAP may be isolated, "Isolated" as used herein refers to PAP that has been separated, removed, recovered, and/or the life© from one or more components io its natural environment, in some embodiments the isolated PAP may have a purity of greater than about 60%. a host cell protdn content of less than about 100 ng/mg PAP, sad/or a host oell DNA : content .of less than about 1 0· pg m¾ PAP. In certain enibodiments, the PAP aay · ν» a pur ity of greater liism. about 9 % in furme embodiments, greater than about 95%; aiid. in yei -further embodiments, greater than about 9:5%,

PAP according†© .embodiments of e present Invention may have a specific activity of at least about 20% compared to the specific activity of native PAP from HMu e PAP is obtained ' or derived, in some embodiments, PAP has a specific activity of at least. -about 30%, 40%, 50%, :60¾4 70%, .80%, 90% or me e compared to the specific activity of native PAP from vcMeh the PAP is obtained nt derived. Methods of assaying gnd/or - quantif g measures- of protein and protein, activity and substrate specificity (1¾ β{ ) are - ell kio n to those of skill ia the art and incltide¾ ut. not limited those- described in US, Patent AppiicaiiOirPubiicatio MQ. 2013/031:7197, the con ests of which are incorporated herein by .reference- m its entirety, some embodiments, PAP according to embodi eMs of the present invention, such as a purified hum an PAP. may have a specific activity of about 1 0 to about t.O Umts/m PAP, such as, but not limited o, about 200 to about S0 nmts/mg PAP or about 250 to about .3S0 units/mg PAP. In solution, PAP may have m activity of about ! > 000 to about 8,000 units/mL, such as, but not limited .o, about 3,000 to shout 4000 units/mL or about 4,000 ' to about ¾000 units mL.

Provided heroin: are compositions comprising PAP, the BAP may be obtained according tfl a purification and/or man afaeiurin method of the present invention. The PAP may have purity of greater than about 60% prior to its- addition to the composition, a host ceil protein content of leas than about 100 ng/mg PAP. and/or a host cell UNA content of loss that, about 100 pg/rng PAP. As those of skill m the art will recognize, after addition to a composition the purity o PAP may decrease if other components are present in the composition. In some embodiments,: the PAP has a purity of greate ifmn about 75%; in certain embodiments, greater than about 90%; in further embodiments, greater than about 95%; and in yet iurthcr embodiments, greyer than aboni 95% prior to the addition to the composition.

A composition comprising PAP according to embodiments of the present invention may comprise PA in a range of about 10 units/mL to about 20,000. units/rnL. k some eiobodhnejits, PAP may be present a composition of the present invention in a r ge of about 1.00 units/L to ebo t !0 ? 0Q0 umts/mL, about 1,060 nnits L to about 10,000 units/mL, about 4.000 uniis/L to about 5,000 units rnt, about 2,500 units I to about 7,500 units/at, about 900 units L to -about 4,000 units/mL, about 500 units/L to about 4,500 units/rnL, about 500 imiis/L to shorn 1,000 imits/mL, about 700 amts/L to about f lOO u&½L; about .50 units/L to about 501 ) onksmiL, or about 100 unirs/I, about 500 units¾L,

A pharmaceutical composition, may be provided according to some embodiments of the present invention, in soinfe em odiment s s a >harma«e¼! al composition of the present invention m y comprise RAP artfi a pharmaceutically acceptable carrier. The PA may be obtained according to a uri ficatioii: and/or mgnufooiurifig. iBet od of the presen invention, Arry aubabie pl niateerttica!ly acceptable e sier kn wn to those o skill in the ait ' wa be used in a pharmaceutical composition of the present invention, hi some embodiments,, the pharmaceutical composition comprises PAP ami saline, such as, but not limited ΐο, 0>.9¾) saime aad/o phosphate buffered -saline-. The PAP in the phar aceutical composition may have a parity of greater t¾m about 60% prior to addition to the pharmaceutical composition. ¾ host eel I protein contest of less than- about 100 ngOng PAP, arid/or a host eel l ION content of less than about KM) pgOng PAP. in some enrbodi ents, the PAP has purity of greater thau about 75%; m certain embodiments, greater than about 90%j in further embodiments, greater: than about fSfk nd in yet further embodiments, greater than- about tS!i prior to the additio to the harmaceutical composition,

A pharmaceutkai composition com r sing PAP according to einbodiment f the present invention may comprise PA in a range of about A uniishmL to - about 20,000 units/mf, la some embodirncnia, PAP may be present a pharmaceutics.! composition of the . resort invention in a range of about 100 rmiis/L to about 1:0.000 uniis/m aboat .1 ,000· units/1, to- about 10,000 Units/mL ? about. ,0 0 umits/L to about 5,000 unlfe/mL,. about 2,500 untts/L to about 7 500 nnitsOnL, about .900 units/L to about 4,000 mhts/mL about 500 mi to about 4,500 unitsftnL, about 500 nnits/L to about L000 uaits/mL, about 700: uuits I. to about 1,100 - ' u ts/mL, about 50 /L to about S00 unks/mL or about 100 umis/ί * to about .500 «Biis mL.

A medicament may e: provided according to some embodiments of the present mveutkm. In some embodiments, a medicament of the present invention may comprise PAP and an acceptable carrier. The P P ma be obtained according to a purification and/Or ntasoiaeturing method of. he present invention. Any snii&h!e earner known, to those of sfciU in the art may" bo used in. a me icament of the present invention, in some embodiments, the medicament comprises PAP ant! saline, such as, but not limited to, 0,9% sahne: m w- phosphate buffered saline. The PAP in the medicament: may have a purity of greater ihao about .60% prior to ibirnulati n or addition to the medicament a host cell protein content of less than about 000 ng/fng PAP, and/of a host ceil DMA content of less than about 100 pg/rng PAP. & some etribodimefits;, the PAP has s parity of greater than a½mt 75%; in certain

embodiments, PAP m&y be present, a medicament of the presenHnvention in a range of about 100 umts L to about i 0,000 nits ra ' L, about 1,000 umts L to abou 10,000 urdts/ L. about 4,000 umts/t to about 5 S 000 i« ts/mL f about 2,500· usit$/L to about 7,5 0 ;uniis mL 3 about 900 units L to about 4,000 urais/ L, about 500 mitsfh to about 4,500 unba/mL, about 500 w /L to about | >: 0O0 : UK¾S L, about 700 units/L to about 1,100 uniis/mL, about 50 tupts/L ■to: about 500 unlts/ L, or a out IQQ units/L to about 500 units/mLb

"Pharraaceuboally acceptable carde and "acceptable carrier" as used herein refer to a carrier that is compatible ith other ingredients in Ibe phatnaaeeiiticai composition or medicament and that |s not harmful r ddepMous to the. subject,.. i.e., the earner can be administered to a subject without caiising any undesirable biological effects such as toxicity. The earner may be solid or a liquid, or ' both, and may be formulated with a composition of this invention a¾ a unit-dose Ibfmidstion, which may contain the compQwid pbc. : PAP) in a amount of about 0,01% or 0,5% to about 95% or 99% by weight of the composition.

The on¾)0sitiofis nd: medicaments (be,, formulations) of the invention may optionali comprise medicinal ag.em.up pha maceut c l agents, carriers, adjuvants, dispersing agents, diluents, and the. like.

The compounds of the invention ma be formulated for adniimstration in a: pbarrnaoeoiica! carrier in- accordance with known techniques. See, g,g ^ Rermugipm the Science A Practice of Pharmacy (9 & Ed, 1995). In th manufacture of a pbarmaceiOlcai formnlaboa according to the invention, the compound (inducin , -fb© physiologicafiy acceptable salts thereof} is typically admixed with, inter alia, an acceptable earner. One or mere compounds may be incorporated in the formulations of the invention, bidh may be prepared by any of the well-known techniques of pharmacy.

The formulations of the itrventioa include those suitable for oral, rectal, topical, buccal aub-ingual), vaginal, parenteral -g-, subemaneou¾ inirsnm&cniar includin skeletal ' muscle, cardiac muscle, diaphragm .muscle and smooth muscle, irrradennal, mtravcaous, ifitraperitoneai), topical both skin and muc sa surfaeea, including airway surfaces), trartas¾l,. tmrisden eii.,. intraarticular, intrathecal, and irihaiaiion adrnmistration, adndnistradou to th liver by inusportal . delivery, as ell as direct organ raject»oa. :(tf,g:, iota ike. liver, into the br¾iiifor delivery to t¾e central nervous system.., into the pancreas,, M g tumor or the tissue stm-ounding a tumor). The most suitable route to any given ease wiM depend on. the nature and seventy of the condition bein treated and on. the astut of die particular compound which being used.

la some embodiments:, a phari aceudeal composition and/or i»edkai»ent of the present ½vv arian may be suitable for intratheeai delivery and/or may be administere to a patient in m thereof inira h-ecaUy. la Cer ain embodiments, a pharmaceutical eompositiou and/or medicament of the present invention may be adsriirdstered by intrathecal liijeetiqn- and/or by a pum pr¾vlding : intrathecal delivery.

For Injection, the earner will typically he a !ksiid, such as sterile pyrogen-f ee water, pvregsn^xee phosphste-byffeted saline std avion:, bacteriostatic, - ater, or CREMOPHOK® EUR] {BASF, Parsippany, H J:). Fo other methods of adm nistration* the earner may be either solid or liquid.

For oral administration, the comp und wa be administered in solid d sage forms, such as capsules, tebleis, aftd powders, ot m liquid dosage forms., such as elixirs, symps, arid suspensions. Conwouads may be encap ulated m gelatin capsules together with inactive ingredients and powdered carriers, such, as glucose, lactose, stjorose, ariaitol, starch, cellulose or eeilmose derivatives, magnesium stearate, stearic acid, sodium sacohm^-tako , mag es um earkmate and: the like, gxarnpies of additional: inactive Ing e ients: that may be added t provide desirable color, taste, stability, battering, capacity, dispersion, or other known desirable features are red iron. oxide, silica gel, sod? am: lanryl sulfate, titanium dioxide, edible white mfc arsd the l&e. Similar diltieuis may he used to malce compresse tablets. Both tablets and capsules may foe manufactured as sustained release products to provide for continuous release of medication over a period; of hours. Compressed tablets may be sugar coated or film, coated to m ask any unpleasant taste and protect the tablet from, the atmosphere, or enteric-- coated for the gastrointestinal tract. Liquid dosage forms for oral administration: may contai coloring and flavoring to increase patient acceptance.

Formulations suitabl for buccal {s bdinpait admiidstration include lozenges comprising the compound Id a flavored base, usually sucrose and acacia or tragacanth; aad pastilles composing the compound, in an inert base soeli as gelatin and glycerin or sucrose and. acacia. FontMlaijo s si3it¾bie for parenteral administration c mpos sterile aqueous and nom aqueous Injection sotetos of the compound, which preparations are preferably isotonic, with the blood of the intended recipient These preparations may contain antioxi ants, . uffers, baeterlostats and solutes winch .render the Simulation isotonic with the Mood of the intended recipient -Aqueous and non-aqueous sterile sus ensions nia include suspending agents and thickening, agents. The formviatiohs m¾f be Resented in .»nii\dose or mnlthdose containers, for example sealed ampoules and; viais, a d ay be stored in a. fheeze-drkd (1yophi!i¾edl condi tion requiri rig only the addition of the sterile: liquid carrier, tor exam le, saline or water- for -injection immediately prior to use.

Extgtaporanenns injection solutions and suspensions ma he prepared rom sterile powders, grannies and tablets, of the kind previousl described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile eomposidon comprising a compound of the invention, in a unit dosage form in a sealed comaker. The co pound or salt may be provided in the form of a !yo hJ!izaie which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for imeeiiem thereof into a subject, The unit dosage form typically comprises from about 10 m to about 10 grams of the compound or salt. When ihe compound or salt is substantiall y waterrinsolnble, a s rident amount of emuisifyiug agent which is pharmaceuticall acceptable may he employed in suffi ient - quantity to emulsif the compound or salt in an aqueoua carrier, One such useful emulsifying agent is phosphatidyl choline,

Toxrnuiations. suitable tor rectal administration are preferably presented as mil dose suppositories. These may be repa ed by admixing t e eonrpounrt with, one en more conventional solid carriers,, fm example, cocoa butter, and then shaping; the resulting mixture.

Tormnlations suitable fer topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel spray, aerosol o oil. Carriers which may be used include petroionm jelly, ianoline, po!yetfiyieae glycols , alcohols, transdermal, enhaaiers * and condonations of two or more thereof

pemmlauons suitable for transdermal administration ma ¼ · -presented- ' .as discrete patches adapted to remain in intimate contact wkh the epidermis of the recipient for a prolonged period of time. Formulations suitable fer transdermal administration jriay also be delivered by Iontophoresis f¾¾ for C tunpIe, Tyle, P atm, R s. I:31g (1986} ? which. incorporated, by .reference herein ih its entirety) and typically take the form of an optionally buffered aq aeons solution of the compound, -Suitable formnlatiotrs comprise citrate or bis/iris .buffer (nil 6} or elhanol/waier and contain from 0.1 to ih2 of the compound, The com ound may alternatively be fo vulated for nasal administration or oth rwise adhmiisiered to the lungs of a subject by a y suitable means,. ir. . g.,.am¾iaistered. by an aerosol ¾3js ¾6si n of respirafcie particles comprising the compound, which the subject mha!es. The respiraWe par efes may be liquid or solid, The terra "aerosol" includes any gas-beme suspended phase, which s eapabie of being i abided into the bronchioles or nasal passages. S ecifically, aerosol includes a gss- ' horne suspension of. droplets, as. ma be produced in a mete e dos irihaier or nebulizer, or to a mist sprayer, Aerosol a&o includes a dry powder composition suspende in ai or other earner gas,, which may be delivered by its ilaiioa from an inhaler d.eviee .for example. See Gaader oa & Jones., Drug Delivery to tf Respiratory Tract, Ellis Honvood (1987) . ; Gonda (1990) Critical Revi ws in Therapeutic .Drag Carrier Systems 6 273-313; and Raehuro. ei al, J. Pharmacol Z' ivoL Metk 27:1 3 f9¾), Aerosols of liquid particles comprising the oompott i may be oduced by any suitable means, s e¾ as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer,, as is lcftowft to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729- Aerosols of solid particles comprising the com und may likewise be produce with any solid particulate medkament aerosol generator, By teekiiques fcnowo the pharmaceutical art.

Alternatively, one may administer the compound m a local rather than .systemic rsaaner, for example, in a depot or sustained-release fotmttiauon..

A c0mposM<Mj pharniaceotleai composition, and/or medicament of the present invention may comprise a t!reraps rtieaiiy effective amount of PAP and/or may he minmu e t administer therapeutically effective amount of PAP. As ' used herein, the term "therapeutically effective amount" refers to an amount of PAP according to enmodhiu ts of the present, invention thai elicits, therapeutically useful response m a subject Those skilled in the art will appreciate that the therapeutic effects need pot be, complete or curative, as lon as some benefit: is provided to the subject. In particular embodiments o the present irrverstiom a therapeutically effective amount of PAP of the present invention ay e administered irrtraxhecally. In seme etubodimeu s, intrathecal administration o a composition, pharmaceutical composition, and/or medicament of the present mveruloft may deliver PAP itr an amount of about 30 nits/mL to -about: 1,000 units/ml, or about 100 u&its/rnL to about 500 units mL,

According: to some embo i ents of the present mventiom a composition,, p armace tical composition, ajad/o,r medicament of the present invention may be administered to a subjeei surd or used to treat a subject. Exemplary methods of administering a d pr treating a subject include, but are not limited to, those described in. ITS, Patent No, 2010/0266569, which Is ncorporate terein by reference in lis entirety. For e am le, in some :e bodi¾e|ite, a method of the ese t invention may comprise administering a composition, pharmaceutical composition., and/or mediea ent according to embodiments of the present m eaifen to treat pain n a s ject cystic fibrosis in: a subject, ari i er a disorde cbai¾oteriEed .at least ¼ part by an e cess of lysophospdratid!c ¾c¾ is. subject. In se.me en odrments, a method of ike present invention maycdmprise administering a composition, ph rmaceatical composition, and/or medicament according to embodiments of the presen im«utio» to a sub] set to general© and/or increase the concentration of adenosine in t e swbjeci. In certain embodiments, a raethed of the present inventioi) ma be us d to enerate Siidfor crease the level or concentration of adenosine in a lung, of a subject

The present mveufion finds use in both veterinary and medical applications. Sihtabie subjects of the present invention include, but are not limited to avians and mammals.- The mm "avian'* m used herein includes, hut is no limited to, dueksns, duoks, g e Sj ¾¾&U, turkeys, pheasants. Mites ' {e.g^ ostrich), parrots, parakeets, macaws, caekatiek, canaries, finches, and birds in ova. The term "man nal 4 as usee herein includes, but is not limited to. primates (a.g„ simians and humans), non-tmman ria cs (Ag;, monkeys * baboons, chimpanzees, gorillas}, boyines, o ines,, eaprines, un ulates, poreines, eqra s felines, canines, lagomerphs, pinnipeds., rodents ' (e,g. x rate, hamsters, and mice},, and mammals in vtero. in some embodiments of the present invention-tie subject is a mamma! and in certain enibodiraents the subject s a human. Human subjects include both males and females of ail ages including fetal, neonatal, infant, juvenile, adolescent, adult an geriatric subjects as sh as pregnant -subjects..

in seriate embodiments of the present i vention, the subject is "in seed of a method of the- present mventiom e,g,, the subject is in pain, i is believed, that the subject will be in pain, and/or it ia believed that the subject is in pain, further embodiments of the present nventio may pro vide a method of producing recombi nant PAP. The recombinant PAP may be human PAP and/or bovine PAP. in some embodiments, a method of producing PAP may comprise cuituring; or growing a cell such as, but not limited to, a mammalian eelf expressing a recombinant PAP, and maintainin the ceils to thereby produce PAP. The ceils may thus be best eeik -expressing, recombinant PAP ( .a,, PAP host cells), in some embodiments, a plurality of PAP host ceils may be cultured.

In some embodiments, the cells are human cells or Chinese hamster ovar cells. Xn certain embodiments, the : cells stably express soluble PAP, Methods and vectors i»r genetically engineering cells and Or cell lines to express a protein of interest are well known

- ~ to tho e skilled m the art; dor example, various tec niq es are illustrated Current Protocols in Mokeuiar Bioiogy, Ansnbel et al, > eds. ( Wiley & Sons, New York, _38S S and quarterly updates} ma Samhroofc et ah, M ½ ¾¾?- Ciamng: A Labo tary Mamat (CMd Spring laboratory Press, 1989) and are described n LIS. Patent Ή 7,D91,M4 and 8,512,983, the contents o each of which: are incorporated herein by reference in their entirely.

The ee¾ i ay be eirftmed or grown in any suitable cell culture medinrm la some embodiment^ the eeii imy bo cultured hi a defined seruni-iree base medium durin a growth phase. The base medium may be animal component, free. ' Exemplary defined serum- · ireg base media mciu ¾ bat are not limited to, toss described in J&blt Π and. those described VI, Patent Mo. 7,091, 004, the contents of wh&h are incorporated: herein by teteren.ee in its entirety.

One or more nutrients and/or supplements may be added to the base medium. Exemplary nutrients a!id/ r st¾ kmenis include, but sre not limited to, vitamins, minerals, salts, gr.o h factors, carbohydrates, proteins, serums, amino- aelds, attaetenent ac ors, cytokines,, -feorrrtones, antibiotics, therapeutic agents, buffers, L-giutamine, sodium hyppxarrthine, rhyrnidke, etc. In some embo iments, the base medi m comprises at least one L-gl»t^in# > sodium¾y^&Xtot &e > and thymidine.

The method of roducing PAP may comprise ma aining the sells- in the base medium during production phase. The cells may be maintained hy supplementing the base medium with a defined -serum-free feed medium. The feed rned nm ma be animal component free. Exemplary defined serum -iree base .media include, but are not limited to, those described in TaBIe 1%.

The ctthuring and/or maintammg step of a method of the present invention may be carried out In a sterile environment using equipment and methods teo n to those of skill in the int. I some embodiments, the temperature or the cuhuring: and/ or maintairting environment ma be about 2 ' ' G to about 4 FC 4 such. as. but :not limited to, about 3G C C to about 40 C C or abou 35°C to about 0¾·;. In particular embodiments, the temperature of the enduring and/or aintaining: en vkomnent may be about 37 " C.

The enituring and/on .maintaining environment mny be m atmospheric -. pressure, reduced pressure (e.g., ¾aennmi ed pressure), high pressure, arid/or any combination thereoC In particular embodiments.,, the pressttte of die euituring and/or maintaining step environment may be at atmospheric pressure.

in some etuborlimeuts. the culmriag and/or mniotaifiing atop may be carried out in an atmosphere of. about 1 % to about 20% carbon dioxide (CDs), such as, but not limited to, about % to about 10% or about 1% md -about 10%€¾.- Ή certain embodiments the euhurmg aad or maiaiairsing step may be earned out ia an atmosphere of about 5% C<¾. Other gases, sueh as, hat ao ' t limited o, nitrogen and/or oxygen, ma be added to the en!turing m&or m Mdmug atmosphere. In some em odiments, one or mere gases may be, used to obtain and/or maintain the desired atmosphere e.g., o: ma nt in the desired o ygen and/or carbon dioxide levels).

The eeli culture may have a dissolved oxygen content of at least about 10%, such as, - n.et limited to, at least shorn 20%, 30%, 40%,. 50%, S0%, 70%, 80%, 90% or mom in some embodiments* the cell culture may have a dissolved oxygen, content of at least about 50%. The cell culture may have a pK of about 5 to about 9, such ' as, but not limited to, about •6 to about 8 ' or about 7,

According to some embodiments, the eelt euit re niay be maintained and/or kept at a ' temperature of about 37°C, have apH of about 7.0, and/or have dissolved oxygon sexvteni: of at least about 50%,

The supplementing step ¾ay be earned out m a eell dmsity of at least, about Id) x 10 & ,. sueb as, but not !feniied to, at least about 2.0 x I f 3.0 t id , 4.0 x W 5.0 x ϊθ\ &0 x 10 s :, 7,0 x U>VS,0 % , 9,0 x lOy or more. In seine embodiments, the supplementing step may he earned out at a. cell density of shout 6,0 x 10 6 ;

A su lementing step may earned out and/or repeated us needed dating a maintaining step. In some en bodunems-, the supplementing step may be earned out and/or repeated two or more times during the maintaining step, such as, but not limited to, three, iow, five, six,, seven, eight, o more times daring a maintaining step. A. supplementing step may oecur shout ever? 6 :hours, 12 Honrs . , or 24 hours or about every other day, every ¾ 4, 5 : , ,or ¾ days, or about once .a week, ia some em odiments, a anpplenren dog step may be cairied out auditor repeated about ewx other day.

A srrppieroentiiig step ma compris adding the feed medium as a botes, at a rate of at least about 1 % of the initial cohere volume per feed: addftioa In- iie-e¾k¾ ii 6n¾S,.iiie-i¾te may be ab®ut !%;, 2%, J 4%\ 5%, 6%, 7% > 8%, 9%, 10%, 11 %, 12%, :13¾' ¾ 14 , 15%, 16%, i 7%, 18%, 1 %, 20%, or more of tbe initial culture vohuue per feed addition.

in some embodiments, a maintaining step comprises supp!eraer ing the base medium with a. iS iga , sued, as, but not limited to, glucose, lie sugar may be added: to the base medium to maintain a desired .sugar concentration, such as, but not Kralted to, a sugar concentration of at least- about I g sugar per L, ¾ some embodiments, a sugar is added to the base medium to maintain a sugar concentration of about 1 : g to about 2.0 g ; sugar jj&r L ar about 3 g to about€ g of sugar pet L< in certain embodiments * the stigar may be glucose.

The cell c ltti comprising PAP may ftsVt PAP titers or a PAP concentration of at least about 50 g L, in some embodiments, the PAP li ers ma be at least abom 100 mg/L or more, such as, but riot limited to, about 15% 200, 250, 3(30; s 350, 400 or more mg/L. In certain e bedimeiife, the cell culture comprismg PAP may have PA titers of about 200 mg/L ¾ about 3Q0 mg/L. As those of skill m the art will :reeogrji2e, PAP titers and/or tbe concentration of RAP may be measures different!;? and/or may jjyov.de· different vamea when me PAP is secretory compared to tr nsmembrane.

PAP may be obtained i n the ceil culture according to methods known to those of sidtf IH the. art. differe t methods may be as.ed if tbe desired PAP k secretory PAP or transmemb ane PAP, For example, whea 1¾e PAP is secretory,, the cellcuiture medium, may be collected and PAP may .be recovered or isolated from the cell culture medium * m some emb di¾ems, secretory PAP : ¾rod¾eed according to method of preseui inventus is separated fs¾m the ae cthiure medium accordiag to embodiments of the present veMieit, Secretory- PAP say also be recovered from host cell iysates. Transmembrane PAP ma be okame by leasing: PAP from the mem¾m¾e, vyfcieli : may be doii by ysing; a smtable detergent solution (e.g., T¾toti-X 100) or by enzymatic cleavage.

The present irrvsmion is exp!amed in greater detail in the following non-Hm ng Examples, xample 1

Clarified harvests .from S L btoreactors {Brx 8 and 9} were prepared .from cultures of Chinese hamster ovary (CHO) cells expressing humao, secretory PAP ¾PAP), Brx 8 and 9 contained cell eidtisre of approximately 4 L of CHO harvest, and were each processed, for clarification, buffer exchange,, and eo Ce tatioiL The detailed steps are:

•t T e Imrvested CliO ceils; were clarified using a single Milbpore POD depth filter, CO!-Kl, 0.054 m 5 , at a ratio of 83 liters of harvest / of depth filter area.

2. The clarified harvests were theft oeneenirated to 0.4S ami 0,.46L fm Brx S and ' 9; respsehvely,

3» Each clarified harvest was buffer exchanged versos 10 volumes of 20 ffiM Tris- Pell Icon- 2 m ni 30 K PES e htape, : The concentrated sample was sterile filt red using 0,22 um Steripak G:P 20 .filter.

The peresfitage- .recovery ef liPAP from both hioreaetor samples for the clarification and OF/OF ' steps was approximately 1(30% by analysing the hFAP activity. The hPAP was concentrated and buffer exchanged to appropriate conditions (20 mM Ttis, pff 7.5) for .ftutter . p¾nficatio» process steps.

Example ' !

Approximately 428 ¾L (Brx 8) and approximately 455 mL (Brx 9) of clarified harvest of hPAP (total ~ 1,150 mg hPAP for Brx 8, and - 793 nig of .h ' PAP for Brx 9) was loaded oa¾ a 2S0mL-ESH lJHQ® Q column. The ESH IJRO® Q coiurrrfi was. previously sanitised with I H NaOH nd eqoiltbr led with 5 coltsrna valttmes (CV) : of 23 mM ttf*«C¾ pH 7.5, Afer ioading the sample, the column was washed with 8 CV of 2$ j¾ Tns-H(¾ 0.Q85 NaCh pB 7.5. Then, proteins containing hPAP were dated by using 25 rnM Tris» HCt 0.21 NaCh pB 7.5 (8 CV for Brx 8 and 5 CV for Brx 9). Three CV of 25 mM I ns- HCf i Na£h pH 715 was used to str p: oft protems otted tightly to the eoJym L All fractions were collected and the flow rate was 50 mL½k or a linear flow rats of 150 emfoour. Load:, flow through- wash, i eJ.uli.on sairrples were analyzed by SDS-PAGE, hPAP activity assay, HCP, and rDNA analysis;.

Approximately 61 % (Brx 8} and 8S% (Brx 9) of the loaded. hPAP activity units wer@ : recovered it* the oration peak fractions. Less titan 5% hPAP activity anils were recovered in the: flow through .fractions (Tables 1 and ¾, No significant activity was recove ed in the wash, or strip .fractions,. There was about 62% (Brx 8) and 45% (Brx 9) host cell protein (HOP) redaction in the sbmm peak tractions wit the final fiCP concentration of 4,948,389 (Brx 8) d 6,772,213 (Brx 9) ng.of HGP /rag of hPAP. The rDNA remained in . -the elation was 2.1 (Brx ¾ and .3,8 (Brx 9) pghng of bPAPl While not ishing to he botmd to any particula theory, the SBS-BAGE analysis of the chromatographic fractions suggests thai the hPAP has been capt red and the data suggest that Eshrtmtto Q anion cinematography using step gradient einiion at neatral pE works well fgt the initial purification. of PA from CHO ceil euKnre,

Elation 7I?S. 64% 3.*· i0 ! 62%

Elution- 1594 i 7¾ 6- I T" ei.xi * 45% 7.Κ "" "

exchange chromatograph resin from Brx 8: and 9.

Approximately 900 ml, (Brx 8) and approximately 345 roL (Brx ¾ of e iaie f om a ESHMUMO® Q column (total - 550- mg hPAP for Brx ¾ arid - 398 mg oThPAP J r Brx. 9) was diluted: 5 fold with 25 wM NaOAc, pH 5 and tea treated with }% Triton X-100 for viral .¾¾eti a i0si for I hour at oom te^era ure. The samples were: then loaded oato a :¾GmL^S.H lJNO® CPX column. The BSiTMltNO® CPX column: was jptevioraiy satutifzed Λ 1 N NaOH and equilibrated with 5€¥ of 5 mi .MgQAc, pH 4.5, After toadtog the sample, the. column was; was ed with 12 GV of 50 mM 1%0:Ac, 0.15 M : NaCh, pH 4,5; Then, proteins : e ¾aimng hPAP er : -eluted- with S CV of 50 mM PJaOAs, 1)229 M NaCI, pH 4.5, Five CV of 5Q eM NaOAc, 1 " NaCi pB 4.S was- fused t s rip off proteins yoimi lightly to the colaratt. Ail fractions were collected at a flow rate of 20 laLPrrha r a JiaeHj Sow rate of ISO. era/how. Load, flow through, wash, and 1 & ' ιοη samples were miy d by SBS4PAOE,: hPAP activity assay, HGP, arid rDMA analysis.. A prior experiment ' demonstrated that these, coridifeoas rea¾0 " ved the majority of prstein imp.ttri t es with 65-85% hPAP reco-ve y.

Approximaiefy 8.5 % :(B rx 8) a 65¾> (Brx 9) of the loaded hP A? activity ¾nits were reeovered is the ekiiipn peak fraetiops (Tables 3 and 4% consistent with t¾e prior f^s≠mmt Mo sigaifieaijl aeiivfty was rgcovered : in tho flo through, wash: or strip if action, There was: afeoui 99%¾ (Brx 8) an 96¾ (Brx 9) HCP reduction the cPitiofi peak .f aeirom with the

{ Table- 3:■ WMmO® CPA i:k®m:ao|jiSphv (Brx ¾)

Fraeitosis Titer ¥¾&me :f Total ; HCP ■ HCP HCP " % rim.

(rngt) £rsL: I efixym : sPA Avg. Total Avg, HCP ft units recovery reduction toad ' SIP 550 i U¾ ...iiAP . N/A NA

- 3¾ 46A 1 8S¾¾ 5*W 9% 5:8

Example 4:

Ceramic, HydmxyapatP© (CPiT) type ΪΙ 40 ro, x ^xm^ chs ffB≠iy was used to further piaify PPAp. However, pcacwr. to the .mi sd-rrsoik Ciirot¾atograpp¾ -eiattQns &O0i the: Esteuro CPX Cl¾ori5atograp¾y were- Puffer exeh¾j¾ed to 25 mM f!epes, S mM NaPO^ApH 7.0 using a regenerated eel ose 30 PDa -membrane, A sample of load, psrmeate, and retesiate were saved an submitted for PPAP ac!Aity and HC analysis.

Ap roxmatel 87% (Brx &) and PS % (Brx 9) of the loaded hPAP atrdyity usits were: recoverd in- the rstentate ' fractions (Ta es S and No signiEeant activity w¾s : recovered in the permeate ftaaticms. There was ahorA 65% (Brx and &% (Brx 9) HCP teductiui in retetpate fr tii s with the r¾ai HCP c RCSiAtltorr of 42,214 (Brx A) and 155,533 (Btx 9} ig og of .hPAP, W&ile not wishing to: be Pound by any partic ar theory, the results show that ¾ ϋΡ/ΐ>Ρ step Is efileient in P fier exotooge for the next step pyrifieatirju as- well s reffiOViugol ' HCP,

AA Table S: Intermediate lF/DF

Frastioas Titer Total ' % HCF i-iCP HCP % r&NA.

(rag/L) (nit) enzyme hPAP Ayg. Total •Ayg. HCP pg/mg- units recovery SgOTig . redtiOliOiJ

: L d 1511 300 453 j 100% AA0A * ND '

.120% s ' .1/7*10' " Τ··]0 : 65%

320 mL iX.S) and 300 mL (ifex.9j of reieMate from liflTP in 2 i»M HE:PES, 5 ΐάΜ NaP¾, pH 7.0 (total - 43S mg. for Brx 8 and 279 mg for Brx 9 of hPAP) was loaded onto a 31 mL (Brx 8) or a: 1 mL (Brx; 9} GET ^ Tpe if colonn, The CHT Ty e If colurnr was previously san ked' wiife 1 H aCJB and e m&ated with 5 C¥ of Buffer A 25ffi: HEPES, 5 M MaPQ 4s pH 7.0). After leading the ssmipte, the ephrom was washed with S CV ≠ buffe A, A 20 G : gradient of 0-100% o Buffer B (2SmM H1FES, ISGmM. Sodium Phosphate, pH 7:6) was used to efete protein o-wn to the eo mn, Fo«f CY of Ί 0Q¾ Buffer B was ¾sed to stri off protein bmmd tightly to the eolraym. All &e fractions were collected ¾o &e flow rate was 10: mLTmia ίβτχ 8) mi 5 L/m» {&* 94 or a Knear flow iate of ISO era/hanr. .;o flow through plus as , sad elmim samples were, analysed by SDS-TAGfA hPAP activity assay, HCF, aud r N analysis.

Approximately 5%: (Brx 1) and 99% (Brx 9} of the loaded hPAP anlivuy units were reeoversd m the flow through plus, wash ikcipris asd tio significant mount of hPAP was recovered to the ektiosi peak: fractiens: (Ta|fes 7 snci ' .¾), There was about 90% (Brx 8) nd 8:9% ¾ 9) HCP red aion m the flow through plus wash ©actions with the final BOP .concentration of A6¾l and IS A IS ng of BCP&g of ffPAP. The tl5NA t & M in the flew ihredgli pl S was¾ fractions was 6.6 (Brx 8) 5.8 (Brx 9) pg/fng of hPAP, the SDS-PAGE analysis of the efarorn ographie fractions shows, highly purified hPAP m the flow throwgh s- wash ffaciiojis witfe BO detectable eoatamlnaat preteits on the gel. White net ishing to "be 6o«nd by arry particular theory, he data suggest that the CHT type II eivromatograp y is capable of removing protein irapuriiies; and rD A, a» isoIa¾mg highly re hPAP,

-3d-

Blades 1 j ¾!: } 130 6% j 17M9 5 i

Ex ample 5

Phenyl High: Interaction Chrom ography £$ was used to remo e HCP from hPAP, Approximately 400 mL (Brx 8) and approximately 370 ml (Brx 9) of the flow through plus wash fractions from CHT Typo II column (total - 3S9 mg . PAP ftr Brx ¾ 5 s ~ 166 mg of AP tor Brx 9) was diluted % feid wi h 2: M MPO¾ pR 7.5- and then loaded oats a 80mL (Brx 8) or a S3 mL (Brx 9) Pneny] HS eolnmri. The Phony] HS column was previously sanitized with 1 N KaOH and equilibrated with 5 CV of a buffer of i M ¾IIP04 !: |?H 7.5, After loading Pro sa ple, the column, was washed with 5 CV of a bnf&r of 0.56 M K 2 HPC¼, pH $ t . Tfaeri, pmi&ies. containing hPAP were eluigcj by using a buffer of 0.1 M ¾HP0 4; pH 73 f 3 CV. Four CV of a :kii¾r of 25 m Trb-«Cf pH 7.5 was used to stnp off proteins bound tightly to the: column. All fractions ere collected: ¾»d t¾e fkm rate was 15 mL/tnin (Brx 83 and 10 mL/min (Brx 9) o a linear r ow rrde of 175,4- ctii¾opf. Load, fle through, wash, and tlmim samples were analyzed for hPAP activity arid HCP.

Aypproxrmately 87% (Brx 8) and 78% (Brx 9) of the loaded hPAP activity itn¾s wore recovered in the elation peak fraction¾ (Tables 9 and 10), No significant activity was recovered in the flow through fractions, About 5% (Brx S) and 14% (Brx 9} of hPAP activity was detected ikon* the wash a strip fractions .combined; There was about 99% PICP reduction for both reactor samples hi the elation peak, fractions with the final HCP concentration of 45 (Brx 8} and 229 (Brx 9) ng of HCP /mg of hPAP. hile not wishing to be bound by any particula theory,, the data suggest hepyl HS chromatography using step gradient -ebM is efficient for HCP removal roiB hPAP ,

Table i Phccy; H;ah Sab ¾y.iirop t?b?e interadion Cferamatogra hy (Bex S) fraetlsas 1 Titer Vaiacie Total % HCP ~ HCP HGP T¾Tj

< L) enzy e hPAP Avg, I Total Avg. HCP

' 11. units reeoyery ng mL . ξ ' .

972 400 389 A Ν·Λ

250 Ϊ39 3 32 I S-.iM G 3 45* 99%

*Caimlation of this valu Is 3m70QT- 5 ; The QJOOI is the liPAP ¾ : i i)i¾¾Skii¾ri m m to volume fcetesse on dialysis which is consistent with, the sample used for HCP

nalysis.

Talsie l¾ Ph¾iy{ Hi¾h th H jirisg! ibic interaction Chromatography (Brx 9) f fac ions Titer Volume Totai % HCP HCP 1 HC ' % rDNA img/ (rnL) . bPAP Avg. Total Av¾. HCP PS/ m &. L) reduction

" '"'" load """""" 44¾ . 37 m A

99? 130 .. : no 1:28. L 7- S (d i 239* 99%

*CaIcui ation oi 'this value is: 128/0:.55ίΜ29. The 0.55S3 i&the hPAP titer be: pre

adjustment dire to volume. lactase on dialysis which is consistent with the sample used for HCP analysis.

Viral iianoflSratkm of hPAP was evaluated, Appropriately 225 mL (Brx 8) and •approximately 85 mL (Brx 9) of the edition fractions from phenyl HS Column, (total ~- 252 rug hPAP for Brx S, ard ~ 90 mg of hPAP for Brx 9} was loaded onto a Opti Seals 4.0 Capsule as a pre ^ Bltration step unde a pressure of 5 Psi generated by a Ritrogeii streaiR. The: flow rate was 2*3 !Jmm, Tl¾e filtered sample was then loaded onto a \¾¾So:lve ¾o fitter as the viral nano-nitrarion step under a pressure of 30 Psi generate by a n rogen stream. The .flow rate was 2-3 ni/min. Samples of load, after pre- filtration and after viral flitratsoh were submitted for hPAP activit analysis.

Ap ro mately 91% (8rx 8} and S% (Brx ..-9} of the loaded HPAP activity units were recovered after the final viral Steatkm (tables 11 and 12), While not wishing to be bound by any particular ' theory, the result ' s show that the viral filtration step has no detrimental effec on the -protein recovery of hPAP and that this step e.ajy be adapted into . a hPAP purification^ process for viral ciearsSPe.

^Determined by A28Q,

*Deterraiaed by A2f0.

A UF/PF step was performed to buffer exchange PAP samples after viral $ttratki¾ such as described in Example 6. " the step was performed to plaee WAf in the formulation buulr.

Approxtn eiy 190 mL (Brx 8) mi approximately 75 mt (B¾ 9) fcPAP samples after yirai fiitraiiQii concentrated by a ropriately 3 fold. The sericeritraied sample was then buffer exchanged to. 0.9% saline using a regenerated cellulose 30 kDa membrane. A sample -.of ' the- load, permeates, and retemaie were saved and submitted for ' faPA? aefi ity au HCP analysis.

Approximately 91% {8rx S) and 81% (Brx 9) of the leaded hPAP activity units were recovered in the retentais -fractions (Tables 13 aad. 14). Mo sigmficaat activity was recovered in the perrftesis fractions. There ma about 64% (Brx 8} d 63% (Brx 9} HCP reduction ¼ reteptate: fractions with the final HCP eoiicsiTtration of 12 (Brx 8) and 36 {Brx 9 Bg/uig hPAP. These results show that, the final XIF/DF step is ehEefent in buffer exchange ΐ ior uladon bmfcr as well as-removing of HCP.

Example 8

A scalable, CGMP ccmptlsrH >: ied-¾a¾li stirred tank b reactor process: was developed ¾r the o uction of mc©m¾jaoi- tom n PAP (bPAF) using†¾e cell Ί AER2S505. The : liPAP eaneentratioTt t fearvest was: about 200-250 m fiL Ail media ¾scd m ik process were cheodeaSly defined, nimal comportem! .fi¾e ; and available as standard commercial products from a major sup lier t& the bi0pharmaeeuiie industry. ½ the process, the ceil !me is robust and its me!&balie properties: ¾fe ll-suited to bioreactor operation, OxygM supply and pH ■control lequiremens are -m within t e capacity of standard proSa^to bioreacrtor&> ■Natrisat e Bsiinipdo3i rates and wastfe product accumulation are reasonable * The process was developed lit $L. stirred tank Moreaetors .configur d and operated as aie-dO n models of production Moreaetors a¾d is expected to- scale to: produetioa ©ioreaeters 1 without difficulty.

The biofeaetor process began by expanding the: eed train from a single via! of the Master Celt .Bank m Growth Medium fOIbco CD FortiCHG / S iri Gloiaraios) using shake ilask suspension culture. Gestu es were maintained, between a proxima el 0,3 - 3,5 xl0* cel!s/mt with 2-3 ass ges: per week. The seed train was- expanded as a standard culture and nsed directl as the. bioreactor inoei um to provide a seeding deosity of 0,4-1.0 xlQ* celis/rnL.

The initial "volume in the bioreaclbr was 66% of the target final culture: volume, of which 25% was the ioocui m and 75% was Production ¾di¾»ft fGlheo CD For iCHO g rsM. Glotaatine 1% HT Sappfemeit - M Medium ibco CD FortiCHO / 20% GE Aciii¾e A / 2M G¾ AetsFeed ¾ was added as a bolus at ths rate 0 f A t ih® initial eulturo volume per feed addition, " feed medium additions were mado ort a ernate days from- day 3 through 11 , Tfc btoreaetor was harvested on day lA sr when the viability fell bdo 70%, whichever oceared fi st.

Culture conditions were maintained at 27° C s . pi! " 7.0 * ,2. a dissolved o ygm : SG% air sgtyratloii through the -entire biofeaetor i -- -Oxygen md carboa dioxide were delivered i¾ro¾gh. a iargemore sparger to control . dissolved oxy en and pH. Sodi.um bicarbonate (0.SM) as delivered to the sasface of as required to control pM, Air was eardinuously cJiei ^red through the -sparger -¾f s fixed; flow rate of 0,4% vvra. N overlay gassing, as xsed in th -development hioreaetors but may e recommended for production fh& cultures in th oieaetors were harvested and used for downstream and: analytical. ii V^ pm^fti, .ati¾ fei o uction of research sup/plies of purified P&t,

Standard cell culture laboratory eqyiproeut was used ibr the process developraeai, Shafc fiask suspension culture in vented, flat bottom feblerrrneyer ilasks oit orbital platform shakers % CC) 3 l¾ca¼tof§ was the primary euhore mode tor eidiure mair e ance,. exploratory screa ing experimeros. and seed train exparssien.

Bioraactar process ideveiopmefit was done in Sartorfcss A-phis bioreaators with Sarton s; Uni-Vesse!. DL orkmg volotse glass vessels:, Bloreaotoi control was preside b individual Sartor to s A-Plos dereaetor cotrirolkrs. Bioreaeior data .tecs collected and managed by a :rietworked S rterks MFCS SCADA program.. Vessels we e: fitted with st ndar eleciro herrdeal sensors for oxygen, and pH, two pitched-blade impellers, L-sparge a s with Lb mm pores, harvesh and sampling tubes. Head plate ports above the liquid surface- were -used for additions of base, feeds, an cells, Dissolved oxygen coa rol was by del iver oil oxygen to the sparger. Control of pH was " by delivery of carbon dioxide to the sparger and pump delivery of base solution, through a heabl plate port, ' The effective rate of air, oxygen, arid carbors. dioxide delivery was controlled by the duty cycle of individual soieijold valves to ro- ide a pulsed flow of these ¾a.ses,. The 5 L biofeaetor configuration pa ameters are pmvided in Table 15,

~4l~ Table 15; 5 L Bioreaetor Standarti Configuration

45° .Pita ed Biaiie; 7S m ,d ¾ 3 sector shejse btedes

" N umber of IftspeHors 2 (at } ' L;& 3.L working voMme level)

Sparger Des-gi Large are sparge bar (2 x i .65 mm holes)

Overlay (¾s Norte

Sparge Carrier Qas Air

¾r Row Rate Max: 109 U m, Effective; F xed ~, 20 ffiL¾in "

On flo Safe Max;: 500 mL/min; effective; Variable " * 130 Control

COi Flo**' ! ' ".n;c Max; 20 lAttSi;; Effective; Variable ~ pH Control

Dissolved Q 3 Control C½ flow » sparge

«1 ! Co tmi C0 2 to: sparge; 0,5 M NaHC€% to AeadLpJate

Materials

Ail mat rials and supplies, other than- the «e¾ w¾r¾- oammeueia! products, suitable- for, both , research and for GMP prodnefiore Fre-sterihze disposable supplies Shake flasks, pipettes, fitter assemblies, bags, etc.) were used for both tnsiafenaiiee -tod- ex erimental cultures. Sterile solutions of Gibco CD FortiCHO, Gibco L^Qhrtm¾ ne s and Gibco HT Su lem nt solutions- were obtained from !pyiirogen. Acti.Fs.e4 A arid AetiFeed B were obtained fkrm GE / P AA. Feed medium was prepared as 20% AetiFeed A / 2% AetiFeed B diluted into FortiCHO. A research ceil bank (RGB) of hPAP AER285G5 w developed for process development and for preparauorj of a GGMP Master CeU Bank, A development c il batik (DCB) was also re ared and used fo -experiments A8R2.1- through ABR2?. Ceils frorn the Master Cell k (MCE) ere ased for experimeet AER2B sad AER29*

All process development op to ex eriment AER2? was done with cells from the DCS. Cells from ¾e MCB were used for AERlS ami AER2 ; Sta dard laboratory cell culture procedures were used to maintain: eelis as shake flask suspension -cultees for all maixdeoaoce ci tires and screening ex eriments. The; - standard ' gro¾¾h medium for calture r Mtenaaee, seed train e aasiSa, and the base medium fbr hiOreactors was Gfoeo CD FortiCKO / 8mM Gibce L-Obttam e / 1% Gibco HT Supplement

All bmreacior process: development was dope in 5 stirred, tank bioreaetors, The basic eonfiguratio and operating cooditioris ttsed have previously sealed p successfully to 10QL and S00L pmdueiioo bioreaetors. A candidate production cell tag, done- AEK2E5G1 and two b ck-up e-aii dates, . clones , AER284B7 aM AEB2 2BS, were developed, The cell lines were developed ;asiug a Chinese Hamster Ovary (CHO) mammalian cell expression system purchased from hrvitrpgen/Life Technologies. The DMA sequence r r-s PAP V imi 2 (SEQ II NO:i and a C3JBCO® Freedom^ 0044 £«1! line:. development kit were . -selected for CH£ ceil expression. The Freedom™ DG44 cell line is GMP Master Ceil Bank established by

Life Technologies, The GHBCO® pOpt-V£C TM -TQPO® TA e ression, vector containin the DHER ene was used, to : develop the r-shP AP expression veeic

acid anta onist which is actively trampotted into the cell via the folate transporter.

each clone, t he AB1285G5 RGB has been certified sterile (21 CFR 610.12 &. USP <7i> Sterility Tests) and free of: mycoplasma (cidiivab-h and non-cultivable by .FD Mips to Co idep). The tested cell hank is considered the: development ceil hank (DCS) and is qualified. for preparation, of a GMP Master Cell Bank ( CB) and for development of r- sbPAP production processes, - r shPAP;Anal -tic¾l fcfe6<¾

The acvivity of r ^ sh A was measured using the ENZCfIE ® Phosphatase Assay kit (Life Teermo!ojdes, Cat; # BI2020T The Merim reference standard, R4, was produced from material prodoeed in the upstream and downstream, process demonstration campaigns. It, was aliquatedand stored at -WC. Staa ard Methods.

Gel! Cadtttre Procedures:; Standard aseptic technique and ceil culture- ractices weps used to maintain cells as shake flask suspension cultures khoat antibiotics. Cell condition, density, and iability were determined by manual ' hemacytometer 7 Trypan blue exclusion. aad¼ BioRad TC u Automated Cell Counter. t¾e- standard Nova 400 BioA yzer assay panel was sed to monitor nutrient and cataboHte eqncectralioBS. Unless ' -stated otherwise, experiments we«* set up i?om o&lfures recovered from, the DCB,

Standard practices to ensure project security include single use of disposable cell contact supplies, -dedicated media for each project, and no concurrent open culture operations os -multiple projects.

Culture M&inienanse; " lb recover celts Irani frozen stojage, tire viai was- quicfe thawed in a 3 C C water bath. The freeze med um was .removed by dilution of the thawed, cell suspension into 1.0 nrL growth (DIBCO® CD FortiCHO^ / 8mM Gibeo L- eiutamipeX o¾n ' atfugait033. (5 min / 220xg t 1.000 rpro-}. and: suspension of the cell ped t in growth medium to a cell density of 0.3-05 x 1G 6 cel!s/ L in a 125 xnL shake flask.

Mainieuairca cultures and shake flask: experimental -cultures. were- generally maintained as a 30 j»L total Culture volume in 125 ml, sbake flasks. Cultures were maintained at 37 °C 5 5% CO}, m a nondau idified incubator at 125 rprrs. on an orbital platform shaker with: a 0,-75'* stroke. Celfe were passaged every 2-3 days to iriaisiam-a target eel! density range generally between 0J-3- x 10 " cells/mL. Ai higher seed deasides, the ceils grow to densities of 3 -4 x I.Q U cells/raL with rxs observable inipact on culture, properties in subsequent passes, DeaiMiug iirnes of 24-27 bra were typical

Development Cell Sank Preparation: lire production deli- line was frozen as RGB vials. (AER28oGS).and stored i liquid Ha vapor phase.

Seed Train P eparation: During early development work, fesh maintenanc cultures, were prepared from the DCB p opriately aaee a month. Early shake flask screening, xperiments and bioreactor process development used seed: trains started fror the ialniensuce eattures, Bioreaetor process demonstration campaigns 1 and 2 used seed trains started frorn an CB vial for each bioreactor campaign.

The seed train expansion for the demonstration bioreactor fan modeled the number of population doublings required to inoculate li e production bioreaetors f 11- 13 PDL), Seed tralus were expanded in sh ke ask cultures up to- 2 L shake flasks with culture volumes appro maiely 40% of the asprlnal flask volume. Bioreaeior Qp ' eratfeft: The Moreaetors wore eoiu¾nred and operated as s ftic*do A nxo.del5s of fie SOQL production bioreaetors. Configuration p rameters eomrooh to all bioreactor -runs are provided in Table I S, Defeul operating parameters comm n to all bioreactor rttns are provided is Table 16. Experimetttgl changes to the default parameters are descri ed in fee diseassion of tie ap ropriate experiment. All ports and dip t es were fitted wift weldab!e tubing to. make sterile connecTions to the vessel daring the riui ith a fer xui tobirrg weidep The assembled vessel Is autoeiavecl without medium. All additions txvtfte e se medbim s cods, feeds, base, etc,) were muc through sterile welded tubing connections, Ail open preparatory operations were da&z it; a biosafety eabmei. After temperature equilibration and. aii saturation of the medinnp the ox en sensor a¾ calibrated ami the pH .c lib tion, was: cQafirmed

io eactcas %'ere monitored daily for cell density, viability, and metabolite: profile, A daily l& siL sample was taken b fore any additions vy¾re m de to detetmiise cell density, viability, metabolite profile, aacf to check pH and dissoked oxygen sensor calibration. An aseptic analytical sample was clarified by eentrifugation (10 min / 2000xg) and stored at -20 rot assay of product corieeiviraiion alter completion of ¾e bioreae r run. Two 1 mi and one 5 r«L retain sa l s Were :take« oaeh day and stored ¾£ -20 °C, After addition of leed medium and gl taniirje ! a second sample was takefi fee a post-feed metabolite profile,

.for all shake flask experiments ceils were seeded i 12S ml flasks to an appropriate density in growth medium, and other supplements as desired- Medinrrs without L-glu amine. ITT, ox feed supplemetmaioa is referred io as medium" in the text, In ba r rnode experiments, iutther a plem ritaba : with feed nedium, glucose, or glptamipe was done during the. course af the experiment Cultures in £ed~bstch experiments were suppleme e as described below.

All vultures wee incubated: shakng at 125 rpm irs a 37 ¾ 5% CC¼ ir&ubator, m ail experiments, samples of 1 ,2 ml were taken as indicated. Prior to taking; each sample, sterile * was added to the eirltare to correct for aa evaporative loss of about ! mL/day, Eac sample, was assayed impiehiately tor viable cell density, percent cell viability,, and the ova assay p el An & ik analytical isample was clarified by centf-l&gati and stored at CPC tor assay of hPAP coricenfrailonaiter cornplfchori of ifae e)iperiiserii:,

Stnd rd Sbake Flask Ras Medinm Screen, Expt AER21 : The base mediusl screen was a standard batch mode shake flask culture assay to etermine: the baseline hPAP productivity of cells in 13 base nieda (Table If) from 6 differeni ysndors. This assay as used as a reference to eompare productivity to determine which base medium should be used for production in bioreaetors.

■Ml ' b t ; er¾-s5i ptei«eijtfeii -with: 8mM L-C¾uastme aad Is: HT supplement. Glucose .(45%) was added as requiredtio nxamtajn ghieoe e eent! io between 2~6g/L s as measures by the Nova.

Shake asks of each bas med m were set up using; cells from the development ceil bank, Basks were seeded t pass 13 (32 days) torn thaw at 3 x 10 s eelismL n 30 >mh total ol me. Cultures were sampled on days 3, %7., 1 , 12, aid harvested on Da 14.

Feed Medium Screen, ExpA A1R23 ; The feed medium scteen was a l d^b icb mods shake tlask productivity assay, The objective was to improve the perfonnattcs of the base medium by esuiehing it itirfeed mediiim to better raa eb the metabolic properties of the cel !¾>«. Cells were ully adapted to the base medium, but were not adapted to the supplemented media, Twelve combinations of ' bass rnedi m and feed medium were examined according to the matri described in able IS. Cells from the development cell bank were seeded into 30 toL cultures -at pass 4 torn thaw (1G days) at a seed density of 3 x Kf cells/mL.

All base media were supplemented xvith. SmM L-Glntatttine and Ix HT supplement. Gtecose (45% was added to- xtafoiam. glucose coneemraiiou between. 2-6 g/L as measured by tbs Nova,

Cultures were .ssm ied on. days 3, S ? ?,. ·$,. 11, 1¾ 13, and harvested on day 14, or &ea viability was <5i)%, At termination of the culm&, 19 mL of culture was eptieally clarified by cenUiftigattott (10 mm / 20§CkG) and stored at -20°C.

■12 OptiCHO - 15% ' . FeedC !0¾€D Eif!eient Feed C „ . M ,

Feed Schedule Screen, Expi AER.28: The feed schedule screen was a standardised fed-batch snake flask productivity assay. The objective was to identity feed medium and feed interval ooa biTiatiotis thai warrant ¾i¾pf investigation: in the ■■ controlled er ifOBinent. of biofoaetefs:. The design of the feed schedule: screen was iodspendeni of the results of the base -.raed&m screen and incorporates a se arate evaluation of the effect of base medium swp lemerdafton vdisn combined with short interrai (2 day) and long interval (3 day) feed, a ditions A matrix of twelve eornbinaboBS of :rtmitip;e additioo of Oibco EilcieKt FeedC or diluted ActiPeeds m a base: medium of CD-FortlCBO / 8 mM Gluiarnirie / 1% HI at intervals, of two days or three days is used (Table 19). Diluted AoiiFeed is a blend of 20% ActiFeed A said 2% AenFeed B in CD FortiCHO, Glucose was maintained 3g/L not to exceed 6§/t* during the course sf the experiment

Goltures were needed as pss¾ 3 from thaw {& .days-) wife cells fkm:i e MOB at a seed density of 2.-25 x ID 5 cells/Ml, in a 30 ml, culture. Feeds were added to the cultures as defi ed in Table 19. Ho adjustments to the feed volume were made to compensate for the v&temt- of culture removed ¾y .sampling,

Cuihires wer¾ sampled on the days sf feed additioa and hhdai!y ater die last addition until viability was <5G%, At teniamaiion oh the culture, 10 mi, of ci ture was. aseptieally clarified by cemmugstion (10 rnin / 2000xG) and stored ar -20°C. Mk 19: Fs d Schedule Matrix

e v v : o t e c oe aa o e m 3' .

Ped-Baieh Bioreaeior Opcraiiori; e liter fed-batch biareackirs ere used to evaluate and reilne media and feed schedule? selected from the sk ' ake flask screening experiiBffliis, and to prodisce i&ateriaj for purification development. The Moreardors were configured asd operated as seala-do R models of ' the 100 i>.¾ 4 ''' 580L f 0 cticnMcT¾¾£>tors-> Canfigarafion parameters COHSKKM- to all hioreaetor tw$ are provided h Table 15,

Default operating conditions are provided: In Table 14, B&eept fbr pH and: .glucose ζά &ή, the de&u!t cond io^ were used % ' all bioreactor runs. Because there is fi-o eo¾re! operatioii when die process pH is in. the: dead hand . the effective operating pH coatrol polrrt is the upper limit of the dead bap early Iri the cun and then drops to the lowe limit of the dead: band when acid generation becomes .si nificant iuing the tun. When aeid g ne ation and COj removal are m reasonable balance., the pH may be any valine within, the dear! band, ai cose was added as required to maintain the con«e?its¾ti«t5 in ths ¾ g/L mfige,

Bioreaetora were sarftpied daily both before and after addition, of feed niediunn The first sample was assayed iminediatdy for viable cell density, % ceH viability, and the Idov assay panel: As aseptic analytieal sarnpfe was clarified by oeMrlfiigatien (i 0 mm 2000x0) aid -stared at -20- f for assay of product concentration after completion of the bioreactor .run:. The second sample wa used- oa¾> .for the post-feed deierrninstion of the !¾va panel of ^nalytes.

Bioreacior raoa were terminated on. days 1 - 0 S depending oft the ex eriment. Viabilit of all reactors a harvest was over 50%. The i¾l]-co itj6ned. medium w& asepiieally harvested d- transferred t Purification Oevdopmeni for clarification and use in. purifieatiop development and produebop. of research . supplies of purified hPAP,

However, jioivsirppiemeoted HyCeil CHO, ActiCHO and ForiiCHO provided the- best combination of productivity and -euftwe psrtorarartee for a production base medium, in hatch shake flask tess, the hPAP coricerjtraiion at harvest was 71 X 63* nd 4 , i mgL, respectively; firowth rates asd peak cell densities were aroi comparable ior these base media, The FortiGHO . supported 45% more integrated celbdays than f!y Ceil or ActiCHO.

<¼·· described above,, AER21, was rtra m & hatch shake flask culture with glucose sappieme tation d¾rmg: th exper ment The experiment -was set up St cells m the development cell bank 0C& t pass IS from thaw. Tk assay results: are summarised in Tafeie 20. The data in Table 2i have been: sorted by the day 14 hPAP concentration & have a different sequsp.ce of conditions than the Experimental Matrix (Table IS),

The three top conditions fl. ¾ 13) lor hPAP concenitation op day 14 are not weft separated fkaxi the rernammg conditions Table M " ). Of these, conditio 1 $t & higher tharveoad rio 1.3 for iiten however eondnion. 13 outperformed 1 & 6 for culture properties. Condition 1 (f OrhCiHO 15% / :i 5%B;|. rovided the highest peak viable cell densities .of the top conditions 02,6 x i O¾¾L¾ relatively rapid: growth rates (24. i br doubling b e to day 3), and: one of the higher specific productivities, Although the integrated cell days and

~SQ- oa!tisre d¾n¾ion of these iop-produasf conditi ns arc geed, eoPditioft S (PsitiCHQ) as the: best .far iMegrated cell ¾ys f50.¾ peak density ξ\7Λ ¾·. i9¾n¾ mid cdtee durat n, ¾¾ίίε n t wisfehig to be to any particula Sistaft with the . ossible ©xcepltoa. of condition 11 (OptiCHQ /20¾ FeedB} 5 fcejs? ddes not appear to te a otess effbi-mauee: advantage for ither Fes A or eedi-wtleii ^a^&i: 20 :eoBG¾jtmtic»irio OptlGIiX either as mdmdaaf.¾F com ' hmsd additions.

The niitrivnt s X rpetaboMte levels: are generally wdhcoritraRed for ¾ll conditions. Gjaeose was exhausted m most of the flasks: at die time of harvest Giutamin w¾s als exhausted 1» most lk$k¾ feai low Gi amine did not nsgativdy affect growth or ¾ «cii¾%. Lactate, a.mmorua, nnd H ere ^efe t iial!y c©n†^U : e ¾ condiions. Tm sodium eoDsesration is constant lor the entire assay period f½ att oDsidftons a«tl ' confirms- .ifee cojteet volume of water was added to adjust itor s^dawors. The hi sge- in osmolality is presume to esuh fioin C0 ui¾ ¾¾ of glucos a§a# m o aci s,

Tal>lo2ff: Base Medium Scree¾ lesuks

Dats are sorted by the Day 14 Titer; tet e th . bye eiir ^ m Moxi as ίϋ the ox erme^l matrM and fe figures. The produc -eeneentmi i) ¾¾s determined osing aotivity assay. Data are: ft am experiment ABE21 ,

con it ons r cu ture c aracterist cs.:

As descrifcad above, AER23 was ran as a -hatch shahs flask culture with glucose supplementation during the experiment. h experiment as set tip whh cells from the development cell bank (DCB) at pass 4 from thaw. Th assay results are sum rised in Table 21. The data in Table 21 haw been sorted b¾ the da 1 hP. P concentration and have a diile iit seqae ice of eoiiditlons than the Experimental Mat ix,

The thtee to certditions {¾ 3, ) for hf Af concentration on day 14 are ialrJy well separated from the remaking conditions (table 21), Of these, conditious 2 & 3 are higher than condition 7, Condition 2 also provided excellent peak viable cell densities 12,5 x ] 0 s /mL (i.e., I2r5 e6 ,j, relatively rapid growth raiea (29,1 hr doubling time to day 3.)., sa one of the highest specific productivities, Although the integrated cell days aud eulrnre dmntiou of these top-prodiicer conditions: are good- condition 5 was also the best for integrated cell days or culture duration, While not wishing to be bound to any particular theory, wi th the possible exception of condition 10 QpiiCBO +13% FeedB) 5 . there does not appear to b a clear performance advantage for either FeedA or Feed when added at 1:5:% eooeejtraiion, either as individual or Combine additious,

The nutrient and metabolite- levels were generally ell-eoairoJied fo ail conditions, Conditions 1, arid S, howev r, ma have been limited by glucose exhaustica, Olutanune w s also exhausted ip. most flasks, hut low Gichamine did ' not negatively nflecf growth or proSirchvity, Ins ate, am , and≠& excepti fia!ly weil-controlled: m. ail conditions. The sodium concentration is constant for th entire assay period for all conditions m& corr&nrs the eotreet volume of water was added to adjust for evaporation. The chang in osmolality is presumed to result ifom consumption of glucose and annuo acids.

Feed Schedule Bc , Ej pt A£R¾ Mulii-Teed Screes:: .Arooog the- feed, media tested, the diluted AettFeed« provided 1 a gniika¾tiy feigner KPAP yield th n CD Efficient Feed C with the s me feed schedule. Both feed media, showed good culture properties with high eeli densities up to ! § $ \if eells/mf and greater than 140 integrated cell days. Culture -d raiioa (16 days / 80 Q% Viability) was good. Tie difference in product yield is approximately 20% between diluted ActlFeeds and eed C.

The experiment was set up with cells from the. Aerial MCB (Pass 3 Day 8 from thaw) using the feed schedule matrix deimed in Table 1:9

The results are summarised in Tabl .22. it should be nored tha the design of this study varies the volume between feed additions, but the feed schedule is essentially the m - far all coitditiotia (maximal $0% -of ¼ilal volume}.. Doe to the feed schedule difeertees. the. first day of feed ad !doo. ranges, from day 5 to day 7 with all feeds completed by day i.3.

Data ffom:-c0tttfol.c¾itures ' instate- thai i general culture perJbrmstiee parameters of eak VCD srrd ICD is this study were compa able to the Feed Medlurn Sereen study.

di ierenee k the iuft gro th ra k Mieved to refleot acceptable variations in the seed cultures and set up of the experiments, The highest liPAF titers are com arable or improved- to. Jiose of the high production, esndiisoss In tie Feed Msdima Screes experiment ( hffelo

The most striking obser ation is the: benefit of ActfFeed ' supplementahom AM feed medium and feed schedule, combinations which included AetiPeeds had ¾ higher Miyest hPAP eofioenhafic-n than any of the combinations that used CD Efficient Feed C, No other parameter had this perfect, esrfelahoa with productivity. The directly measured culture: parameters (peak VC1 Gidture duration, viability at harvest), i. the derived" enltnre: parameters (doubling time, lOD, speeife prDduehvlty), correlated positively to. product yield in eaiy a general sense. An increase In both ICO and specific productivit were recfuhed to maximize yield.

Ameng the highest yield conditions, the diluted efiFeeds with a standard bi-dsily addition beglmhng day 5 rovided significantly Mghsr product yield and cellular specific productivity than, other samples.

l¾r the fed-hatch conditions. hPAP yields do not appear to be limited by nutrient dxhaustion-or waste Ttieiabo ' Hte-aceu«i«lai¾», G ' iocose CQneentralioas at harvest were greater than 2 g I, a¾d jyaatsniate concentrations were greater than 2.5 aif, the peak lactate nd ammon a concentrationa were sss. th 2 g/IL and: 9 M, respectively, values well below those frequently encountered iu production bioreactors. The midmurn pH ranged fxoro 6.S to 7v0. and the maximum pH was 72 to 7,4. Even though there was BO attempt to actively control pH in this experiment, the R remained wi m a range freque tl used in production bloreactors and suggests pH control will not be difjlcyit to achieve.

The significance of the apparent near exhaustion of giuta ine is .not. dear. Gibco applications: specialists acknowledged that components . is i ike EfSeient Feed Media interf re wit detomnnafion of " ghitatniB© by the e a bioandyzer, hut eoul not deserlbe the nature of the interfereneei Based on HoM bioaxiaiyzer measurements of gmtsnfne he&re and after addition of gkaamirje to cnitures, changes hi concentration m the aiM to & mM range are nan be measured and are relatively accurate. Howeve , it appears the interference causes an elevated baseline at rawer gmtamine eoncentraiions. The }o%v residual concentrations «f gl ramins observed in this experiment may be artifacts of assay imerfere ce,. Although glutaffiiPo consumption was initially rapid, It appeared to reach a stead state and no additional ghiiamuie was added during me experiment 1 is not clear, but the: assays suggest that giutamine is not. required as a nutrient supplement,

The results of the feed sehednle screen, for the most parti appear to he internally : consistent and In reasonable agreement itl¾ the results of the su pleme te miediutn screen. pihfied Actifesds hb 1 8¾Vi ^dally additiba ro ides lk most coasrsteai improve ent m nPAf production &s the feed: medium for fed-fesicb euitwrs< " hi addition, the feed sched le screen d^oas dated thai, supp!smetifatiori of the base insdlwm wit u}su!i¾ does: : ¾:oi enhance fte product yield.

While noi wrahing to be bound to y particular theory, based on both the feed screen. t tie se Ϊ$< ¾ΙΒ¾ ,sc«w &$u¾s, :$t£ most: Kmsismg feed medium is diluted Άοΐΐ Feeds, Further, titog wishing » b&iwd to my- psjituclar theory, the most promising feed sche le is t supptetnetit wih 50% (V?) feedtnedi rn and to ad feed riicdiam a ibe rate of 10% (vv) per addition on. a 2 day interval starting when, cells are ½ maximal cell ensit hich: is iypiedly around day

Example 9

A proeess fm pr»duemg . reeomfeh ai human PAP fiPAP) was peformed on the. FIarea<M.or scale with tFde separate hioreactors (Brx 1-9}, Reibrerscs h rnade to equipmertt, . terials, procedures, data, etc, in Example E!ampuign. ΐ (bfereactyrs F 2 » ¾ d 4) and caiii aig« 2 (bioreactox 5, 6, and 7) were process develo ment cair^ai ns which explored aad refitted fed-batch strategies: suggested by the slices flask s£re¾n¾ig. studies. Campaign 3 loreactors " 8· and 9) was a process emonstation campaign to demosstrgte process een¾steney and. readiness for s e-up modrntim, and to produce strpplies trt demoasiradoii of the pudiacatiori process; Ail bioKactors: were harvested and ased i¾s piirBcsiiOij and assay development.

he feed schedules are proided in Table 23, Ail base 5¾edia were su plement d tih8 .t-gteteke a 1% HT $u^leme»t !

PortiCMO Oil AedFfe s 10 3, % % % tt, n id bioreaci u seed trais etaila are provided m T¾Me .¼· The seed trains ihr hioreaomr Campaigns 1 arsci a were ifesved ircrn the Beve!opmeM CeH Bank CB}.: ard the seed trier* fo Campaign.3 ws, thawed from, lie Master Gel! Bank ( CB). FM Id is the oubling iiBjts ihrs) of the last pass beJhre meci;dahe¾ of de bioreaelor, Average Td is the oubling ime, is cslealaisd b di iding th elapsed ime from tha te bica-eacrtor m cufetioK fey the PDL S¾m ik of the ank. InocuittiR VC and % V .¾ the Inosu aa eeliare riMiedi ely before, irjocdaiion of the of c-toi, Brx Seed VCD is the eel! density measured hi the hioreactor iffimediatel after fcoea¾tieh;. Ί , fciofsaetor perfbraafice is rodded la able 25,

urn B 15 25

The kPAP seed train expansion: -and: fed-ba& r bksreactoj- process t¾>r ea¾li campa gn were car ied i as described beicrw, WBere multiple criteria are provided, they≠&s believed to be eie iigeabje and are provided to grv¾ flexibility in designing the mairufiictiiring record. Materials:

Cox o :Manuiacturer Cat. ¾

0JBCO €D ForfiQiQ™ inviirogeii A1148301

Giboo Gl tamine lovitrogen 25 30-081

HT Supplement laGtrog n ! 1067-030

AetlCHO Feed A, liquid ΡΛΛ / GB Ut 5-072

AetiCHO Fesd B Jioidd ¾%A/GE UO5-0S

SAJC GS769

SodnjfH Bicarbonate, 7,5% SAFC $876!

Seed Train Growth Medium:

CD ForiiCHO / ginM ilusmim

Qty / 1 L Medium

CIBCO® CD ForiiCliO™ 960,6 ml.

Gi eo: GMta in 40,0 ml

Produetiori Medium:

CD FortiCBO / 8mM akfa wae i¾ v/v HT Supplement

CoiHpoucai ViA!S L Vol /5 L Vol/ 00 I.

Brx Brx Brx

GiBCO® CD ForiiCHO™ 2360 mL 23,60 I, * 236. 1

Gibco Gtuismine 99 mi. 9.99 L 9,9 I

Gibco FIT Sw piefiieiii \%. 24 mL Q.24L " '" 2.4 L "

Production Feed Medium (Diluted AeiiFeed):

CD ForiiCHO / 20% AciiFeed A v/v 2% AciiPeed B V

Vol SLBrx Vol / 501, Vol / 500 L '

Br Brx

GIBCO® CD FortiCHO^ 1290 ml .....l¾g.L 129.0 L

GB/ BAA .ActiFeed A 33 nil 3,3 L .33 I,

GE/PA -CiiFeedB 33mD L 0.33 L 3, 3 L

Seed Tmm Ex nses;

Thaw rapidly, diMe q iofely with- Growth Medium cen¾i¾e 3 . esuspend in 30: mL Seed team Medium and place in & fist based shake flask. 33xpaftdflask(s) every two t three days.

Seed Density 0.2 - 9,5 lO'VaL

Split Density ! ,5 - 6. -x 10¾mL

Growth Rate ~2SG0 r average doubling time over expansion from thaw to bioisactOTSeed.

Expansion D raiion .18 days for SOOL oreastor vom e

incubator 37 B C/5%C0 2 156 tpm for 125 mL shake flask

130 tpm for 500 n L shake ilasks

1 15 rpm for 1000 mL shake flasks

1 OO.rpm for 2000 ml, shake flasks.

Fed Jkteh Bioreaetor Set Op:;

Sp ge? Large Btibbls; Sparge te with drilled holes

WV 93% of MaXtmurn bio-reactor -workin volume (46SL for 5001, bioreaetor)

Vi 71% of WV ~ Initial set up voiuras afte addition of inocidiim (330L ibr 500L ■Moreacior}-

P xctiap Base edlwrn 75% Vi (24,8 L) (248.0L)

feoc lum Vo ume 2S% 8.2 L 82 L

!irioca!iirfl Cell Density 4.0 6.0 x 10* edisteL (5.0 x l ( f ccils/rnL preferred) hiocrdum % ' V lability >9S¾

inoculum Qrs-w& aie Td 23-27 ftr is expected

Bocultim Populatiori Doublings predict 1 1 PBL to seed SOL bioreaetor a 1.0 1 ¾ ceiis L

predict. IS PDL to seed SOOL bioreaetor at .0 x 1 , ' Q 6 cel L

Seeding Density 0,7 x ! 0 s ceils/mi target (PD has seedsdat 0.3- 1 ,0 χ .10" cdis mL.)

Feed Rate Schedule (If d eds begin on Day 4)

: €attiire l J 4 ύ 8 j 10 12 1.4

f ed Kate 1 .. , l/ i 0% 10% 1 io% 10% 10%

' Glucose Feed As- required to maintain 3-6 g/L; by daily Hova assay (estimate 1 for

/ 17L far S OL bioreaetor;: eeusumption Days 6 ; 8, 10, l and 15 in .PD Heriro Rea KTi)

Harvest Day l b or 70% vi¾ Hty, whichever is: first

Bioreactor Culture Properties;

The cells are generally robust and easy to maitstam ' ia culture. Growth rate is abo nd 2S-3 ' 0 hour doubling times iMoa Hoiii the seed train. Ceils were th ed into a 12SmL shake itask in 30rnL medium using: the s ndar thaw procedure and allowed to inenbate tor 4$ hours before t a first passage, Following passage 1, ibe cells were e panded every two: to. three days until the hioreactof inoculation.

The model for this process description is PD Brx8. The maximal density observed .in Brx6 was -145 5 x 10¾BL on days 7 through 1 L Viability was. very high, |Q~99¾, throughout culture . Dilated AetiPeed was deli vexed slowly (S mL/min) to the Ft ) hloreaetors on days 3, 5, ?y¾ Π, and 13,

Glucose was added n days 4,. 8, 10, 13 . , an : 15, While not wishing to be bound to any particular theory, based on he PD bioreactor, it may Be possible thai a fixed a tiort of 31 of 45% glucose on non-deed days will maintain glucose In the appropriate range in the SOOL bioreacior>

Base addition requirements fer ρΐΐ corr rol were very le>w an the FD bioreactor due to low lactate concepiraiioaa, Om 20mL 7.5% Sodktm bicaraonate was added during the entire run. Lactate is; not fully consumed at any point in ike run asd pH values maintain within the wide pH copiyo band,

i the bioreastof, oxygen consumption was low fer the first 5-6 days of th culture thm pe ked at around op da 6. In PD S oxygen demand fo BrxS reached -500 niL mm m day 6, Oxygen demand emained around ί)0-5δΟΐηίΑ¾ίΐι for the remainder of the culture. While- not wishing to be bound to any particular theory, ox en transfer is n t believed to be a problem in the manuikemring bioreaeiors. Campaign 1 - Bktfeactors 1, 2, % *¾ 4, Expt AER2S

C¾a¾|¾| 8 1 evaluated ied-hatch. ¾k>rea«tor performance using fou strategies suggested by the shake -iasfe screenin experiments described In Example. 8. Tliree base media (CD FortiCEO, ActiCHO P, and HyCell CHO) were compared using CD Efficient EeedC, ActiCHO P w th diluted ActiFee ' ds showed promise in the shake flasks * so it was included m Bioreaetor Campaign 1. The &f msm & was eomparabfc in some respects. Product yte¾, cell yield, and i¾ei¾h$ic behavior were excellent, hu differ significantly between the base media. ActiCHO m ForuCHO were selected &r further development becaus there was evidenc suggesting greater potential for productivity than HyCell CHO,

Biereaeters 1 , 2, 3, s 4 evaluated three iffe ent base media as well as different feed strategies for ActiCHO.. ' These conditions were selected became they provided; the highest hPAP y M or best culture conditions in the Feed Schedule screen, HyCe!i CHO was included in campaign because it slo ed such high, specific productivity in the. feed medium: screen. FortlCHO had the best culture characteristics of any medium tested. ActiCHO P has .shown the. highest titers: even though cell densit and ICB values had been fewer than FtetiCHO.

The default operating conditions, were ' used far both bioreaetors. The seed train and fed-batoh hioreactor operation were performed as described above. The default pH set point of 7.00 ±0.2 was ased beeanse the efieetive control range of 6,8 ~ 7.3. avoids the pH extremes seen the birke flask cultures (6.6 to 7,5). nd is consistent with ranges commonly used for production.

Cell :density 5 viability* -and growth rate was similar in. both bitireaetors until da 3, after which growth la Btoreacfcor X and 2 slowed in comparison i© hioreavtors 3 and 4- Bioreactor 1 reached peak cell density at day 1 1 , which, was later than the others (Brx 2 ~ day 10, Brx - day? . , Brx 4 - day ). However, the peak cell density of Brx 1 was me highest of the rou (17,3 x 10¾elfe inL vs. Brx 2,, 3, and 4 which, had peak densities of 11.6 .%. 1 , 13.2 id 6 , and 12.5 10¼HsAmL respectively). The cell density hit plateaus in Bioreaetors2, 3, and 4 after the feeds began white in Bioreactor I the cells continued to grow slowly up to a day 10 peak, Bioreaetors were harvested on days- 14 (B x3 r IS {Brx2) 5 16 (Brx!), and 1? (Brx4) based on viability (¾80%) to facilitate eon arison, avoi protein degradation b proteases released ftom dying cells,, and to provide material to begin purification development.

rjifferences between the Bioreaetors in integrated ceil days : and product yield were as much as two-fold. Bloreactor 2 had approximatel 20% higher hPAP concentration at h thaxi Bioreacio 1 ;: eh was the second highest fouv bioi¾a?;fQTS sho ed a cOHstait crease in.hPAP coriceohaioa hmugliout the run. Wife iiot wishing to he. omi, to an particular theor ^ there does seem to Be ¾¾. ioMbitory effect n cell .growth with the use af CD Efficient FeedC as. is evidenced by Bioreactor 1 continuing to grew for so. additional 5: days after feeds hegaa compared la (he other three bioreacors hose growth rates slowed sigpffieaatly at iiis time..

:parat»eter was clearly em-related with the irriproved cell densities.

" ohbliog times at day I for bioreaetors,. day 3 for ' shake flasks. Campaign 2 - Btoreaetars 5, 6 f &. 7 : AER27

The obje<r£ive for Bioreactor Development Campaj ft 2 was to eo -mae exploration of base / feed combinations m the controlled en ir nment of a btofeacior.. AetiCHO provided nice level of prodsietivily in Campaign. 1, but the growth characteristics provided by FortlCHO wme enough not to omit ft from eoMldera ion, In Carttpaiga 2, AetiGKO continued to prodnee well whh titers in the range of 250 g/L for both feed strategies, Ilo even PoriiCBD with diluted AchFeeds ioreactor 7) achieved a harvest die? of almost 290 mg L,

Ceils for Cainpaign 2 wore ntken fioat the CB and bio eaetors: were seeded at passage 6, 19 ays posi"thaw> Bioreaetors 5 and 6 of Campaign 2 ir ored Bioreaetors I and 2 from Campaign I with a ·! day shift in die feed schedule. Bioreactor ? nsed the same base m dium: as, Bioteaeior 4, but used the dilated AetiPeeds as feed instead of CD EffleieM EeedC. Ail three reactors were seeded at a target seed density of 0.5 x- if Ye ml This slightly higher seed density allowed feeds to begin one day earlier than the previous bioreaeior. cahapatga, Each reactor was fed at ICtvh on days 4, 62 S, 1¾ md 12,

Bioreaetor 7 was superior to 5 and 6 throughout the ran. Brat 7 was the highest for harvest titer (293.ging/L¾ peak denshy (202 s 10¾mi), ICD {!307¾ a d led in specific productivity whit day 12, AeiiCHO + f eedC fBrxh) had highsr specific productivity for die last 4 days of the im.

FeodC again showed growth inhibition as Brxd only reached a peak density of 9,63 x lQ¾al compared to Btx5 which reached Ϊ7 ' % with the same base m dium

(AetiCHO P)i the. low VCD of B xb only allowed it to teach 75,*· integrated ceil days compared to BrxS (118-1 ICD) and Brx? (135.4 ICD).

Metabolites - d pB were wed eontmlled in. all More ctOrs. of Campaign %.. Glneose add tio was only required once for -BtxS and Btx6 and three times for Brx7. None of the cnlhnes: generated a high: level of aarnonhn The : osrooi aiiiy was hi gher in BtxfS than the crffief two, which may explain its highe Specific productivity toward the end of the run.

Campai 3 - Bioreaetors: 8 k 9, Expt ABR29

Bioreaetors 8 and were iw with identical parameters: to replicate- the planned fed- hatch ocess of development Bioreaetor 3. The seed train sod bio eacior operating csnditions were as; described above. Glucose was maintained at 3 - hg/L by su lement tion as reqiMed. Cy was seeded at 0,1 x Bn Ve ral with a target of 1 %■ c v¾t¾ the objective of drivin the titer higher by starting ith mo ceils, The biorsactors were harveste at Day 16 with viability still high at approximately 85% tor both reactors.

Bkrreaaor 8 peaked at 16 x )0 6 ee!ls/ml on day 11 and then declined slowly. Viability was -stabj - ' Ηφ. ' through ut' ' the ra slowly declining alter day 12 arid was harvested at 85,6%, Biorrjsct r 9 peaked a %M x. .l6 6 c M on day 10 arid then de hted Slowly, Viability was stable arid high throughout the run slowly declining afte day 12 and was harvested at 85.1%. Bioreaetor 9 had a dissolved oxygen (1)0) probe- ailure on day $ and was unable to control gas ¾i¾ure for several, ho rs. This failure as repaired and the culture continued growing but experienced a lag in ceil density wt pat-&£ to Bioreaetor § where the density was about 20% lower k than in 8.

Pr d¾ei titer peaked at 245.2 rng L on day 16 (harvest day) ίοτ bioreaetor 8. Bioreaetor 9 reached a titer of 218,3 rag/L at harvest.

Titers were 15-20¼ lower than Brx 7 of Cam a gn 2. While not wishing t ha bound to any particular theory, this difference may be variability etw en assays, but could he the feed medium diluent:, hi Brs % ActiFeeda were diluted in. AedCddO whereas fa Brx's 8 & % feeds, were diluted i» FortiCHO, Overall, the eidlures perforrned well with iirnited base addition, not inueh ammonia build-up, and normal osmolalities,

(Mucosa .and glutamaie concentrations were all- Well controlled. Glucose supplementatio was required 4-5 times lor each reaetor when glucose le els^ Mi below 3g/'L as measured by BGA analysis,

litis Campaign demonstrated that the fed-batch process Is stable arid reproducible and that extending the culture past, day 16 does act increase total, yields significantly. While not wishing to be bound to any particular theory, it may he desira le to harvest before viability drops significantly.

Bioreaetor hPAP yields in the demonstration bt ©reactors were consistently in the range of 20(h2S0 mg L at day id, whereas the first development bioreaeiors were up to ' 300rng/L. The ceils, in the demonstration reactors grew at n arby the same ra e as observed in reacto 7 of campaign 2 » . which was the model for the process. The difference- in harvest titer p¾ry be assay variability or due- to the f^d; medium diluent

The process should scale up to a 300L reactor as it was developed ' Using, bioreactors configured and operated as scale-down rnodgis of production bioreactors.

- OH- I¾e toiegoiag is llustrative of the present Invention, and is not to ¾e construed as ¾mUi¾ thereof The myeat oa Is defined by the following claims;, with equivalents of the claims to be included therein:. Ail publications, patent applications, p tents, pateiit publications, sequence identified by GenBank and/or SMP access on numbers, md other references cited herein a incorporated b .reference m i i entireties for all teachmgs r levant to the sentence; and/or paragraph in which the xzS&m is presented.