AU2013224728B2

AU2013224728B2 - Stabilized Compositions of Proteins Having a Free Thiol Moiety

Info

Publication number: AU2013224728B2
Application number: AU2013224728A
Authority: AU
Inventors: Kris Lowe; Vinh Nguyen; Zahra Shahrokh; Gaozhong Zhu
Original assignee: Takeda Pharmaceutical Co Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 2006-02-07
Filing date: 2013-09-06
Publication date: 2016-10-20
Anticipated expiration: 2027-02-06
Also published as: AU2013224728A1

Abstract

Compositions of proteins having free thiols, and methods of making and using such compositions, are described.

Description

STABILIZED COMPOSITIONS OF PROTEINS HAVING A FREE THIOL MOIETY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Australian patent application no. 2007212021, which claims priority to US application serial no. 60/771,555, filed February 7,2006. The disclosure of the prior applications is considered part of (and is incorporated by reference in) the disclosure of this application.

FIELD OF THE INVENTION

The invention relates to compositions of protein having free thiols, and to methods of making and methods of using such compositions. The compositions have optimized stability.

BACKGROUND OF THE INVENTION A drug product (e.g., that contains a protein) can be stored in liquid or lyophilized, i.e., freeze-dried form. A lyophilized drug product is often reconstituted by adding a suitable administration diluent just prior to patient used.

Active protein may be lost as a result of physical instabilities, including denaturation and aggregations, as well as chemical instabilities, including, for example, hydrolysis, deamidation, and oxidation. The stability of a protein drug in a particular form, e.g., in a liquid or in a lyophilized form, can be an important consideration in selection of a product form.

SUMMARY OF THE INVENTION

In general, the invention features a composition which includes a protein having a free thiol (-S-H) (e.g., on a cysteine residue) and/or other moiety subject to oxidation (e.g., Tyr, Trp, or Met moiety) and a carbohydrate, wherein the carbohydrate is present in an amount sufficient to maintain the stability of the protein, and thereby of the composition. In a particularly preferred embodiment, the moiety to be protected is a free thiol.

Compositions and methods desOTbedhdefe provide lor increased shibility and storage life by increasing the stability of a protein ««tamed therein.

Compositions described herein, e.g_, liquid compositions containing a protein, have prolonged stability. E,g,, under preselected conditions; e.g., upon storage ift a gas tight container, at a temperat ure of %$ eC for a peri od o f op to 4 6,9,1 % or 24 mon ths (or in some emixtoimeaits longer}, ά protein in the Composition will retain at least 50,55, $0, tS5,70,15, §0·, 85,-80,:95,99, or 100 % of the stabiliiy'ii had prior to storage.

Stability, as used herd», includes parain«aeis such as protein sbucture (e.g., miniraizing Or preventing changes in protein structure, e,g., protein aggrega{iQn.or protein degradation (e.g.. hngynentaririn)) and/or a biological achy tty of the protein, e,g., the ability to convert substrate into product.

Protein stability can be measured, e.g., by measuring proteto a^regaiion, protein degradation, or levels of a biological activity Of the proieiji. Protein aggregation can.be detennined, e.g., by size exclusion chromatography, non-denaturing: PAGE, or other methods for delenmntog size, etc; For example, the composition can have less than a i, 5.10,15,20,25, 30,35,40,45, of SO % increase to the amount of protein aggregation (e,g.f as measured by size exeluslon diromatographyi as compared to the anuatot of protein ag^egation tMf was itj the composition prior to storage (tags, storage at a teniperdtoto df2>84‘C for a period of up to 3,6,9,12, of 24 months (or longer)), Protein degradation can be deieireined, e.g;, by reverse phase HPLC, non-depatmingPAGE, ion-exchange chromatography, j^pridemappiilg, or similar methods. As im example, the composition can have iessthan a 1,5,10,1.5,. 20,25,30* 35,49, 45, or 50 % increase in the amount of. protein degradation (e.g„ as measured by reverse phase HPLC) as: compared to toe amount of protein degradation that wa? .in the composition pri or to storage (e.g, storage at a temperature of 2-S*C for a period of op to 3,6,9, 12, or 24 months (or longer)). The biological activity of a protein can. be measured, e.g., by in vitro or in vivo assay®, e.g., ELISA teg., to measure binding or enzymatic- activity) and other enzymatic assays <c.g., specftopltotometrie, flnorimetric, calorimetric^ diemiliamnescent. todidmetrfe, or clurOmatogfaphic assaj^), kinase assays, and so: forth. As an eximqr-e, toe composition can have Jess than a 1,5,10,15,20,25, 38,35,411,45, or 50 % decrease in a biological activity of the.prototo.(e,g.., .enzymat£C'aouvjty, e.g.; as

A measured by an in vitro assay) as. compared to the amount of the biological activity that vyasin the compositor prior to storage {e.g., storage at atensperatureOf 2-.8*G for a period: of up to 3» 6, 9.12, or 24 months (orlpnget)).

In one aspect, to protein does not modify, p.g,, clqave, any otto components of the composition.. For example, in one preferred embodiment, in ^composition containing glUcocerebiosidase fGC8), the composition doss not contain polysorbate as a surfactant because GCB can recognise polysorbate. as a substrate and can cleave polysorbate to release free fatty acids.

Embodiments of the inVeitd op have stability ebmptoble to th«I of a lypphilized composition of t he same protein. A liquid composition described henrincan have gt least 5(),:55,60,65, "0,75, St), 85;90,95,99, or 100% of the level ofprotein stability (c;g,, retained activity.) of a iyoidnlized coniposition afterS, 6,12, LB, or 24 months: of storage {e..g„ if a lyophilito composition has retained. 90% of its activity at IB months, the Ctoposiiwsn ofto mveniion.has retained at least 50, 55, 60¾ 65, 70,75, SO,.85,90,95, 99, or 100 % of that level).

In one aspect, the disclosure features a composition that includes a protein baying a.free thiol, sad a cMtohydrate,. wherein tire carbohydrate is present in an amount sufficient to maintain to stability of the ptottoand wherein the pH of the composition is tos:ttoaif7.0, In spine embodiments, thepomposition also includes μη antioxidant, "wherein to antioxidant and carbohydrate.are present in «mounts sufficient to maintain to stability of the protein, and thitoiy df to compoatibn, and wherein to pH: of to catiipositioh is less than 7.0. For example, to antioxidant ia cysteine; cysteine hydroehioride (eystoinedlGi), or methionto (e.g., present at between, about .().001 and about LO % (wi/vQi)) and to carbohydrate is sucrose Or trehalose (e.g., present at between about 1 ami abotii.40 %.{>vVv01)>, Tn certain ;e«ibo®n»etits, file pH is in to range of about 4.5. to about 6.5, e,g;, preferably bjepveen about 5:0 and 6.(3, c.g.,nsorc preferably between about 5,5 and 5.8 (e.g.4 about 5.7j. In a preferred etnbodiruent, the composition inciudesa surfactant (e.g., poloxamsr IBS).

In a preferred embodiijient, to pH pfto eomptotionis;. e.g., between about 4.5 and about 6.$, e.g., between about 5,0 and about 6.0, e.g., between about 5.5 and about 5,8 (e.g., about 5.7).

Ih: certain .smfeoclimenis, the stability is ai least. 5-80 % ^greater (e.g., at least about 5%, .at. least about 10%, at least about 15%,· at least about 20%, at least about .25%, at least about 30%,·at least about 35%, at least about-40%, at teasbabeu) 45%, at least about $0%, at least about at l§as( about .60%, at least about 63%,. at foasf about 70%, at •least, about 75%, or at. least about 80% greater), under pre-sdected conditionSi. than the Stability of a comjMs&ion. which differs by lacking the carbohydrate (and the antioxidant,· If used), in. certain embodiments, the carbohydrate (itud optionally, aa antioxidant) is present In an arnoant softkietii to stabilise the free thiol of the protein (e.g., the protein shows less aggregate formation, &.g., the protein shows about 5%, -about 1 0%. about. 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 9.0% .abo ut :95% or .about 99% less aggregate formation utBler pre-selected conditions than an otherwise identical, protein composition that does not contain the ctabohydrate (and antioxidant, if used)). in «Srtain embodiments* the .carbohydrate (and optionally, an abfienddani) is present in .an amount sufficient to increase the stability of Hie protein (e,g„ the protein shows less ag^regate fomtatioj-i, e.g., the protein shows about 5%, about 10%, about 15%, about 20%, about. 25%, about30%. about 35%yabput40%, about 45%, about.50%, about 55%, jfbout'6Q%, about 65%, about: 7Q%, about 75%, about 80¾% about 85%, about 90% about. 95% bt about 99% less aggregate formation under pre-seiee&d conditions than an otherwise Identical protein composition that- does not contain the carbohydrate (and antioxidant,, if used)). fn certain enibodiments, the carbohydrate (and optionally, m antioxidant) is present in. an amount suffi.dei.rt id inhibit the reaction ofh free thiolon a first molecule of the protein With, a free thiol on a second molecule of the protein to forth an aggregate.

In certain embodiments, the carbohydrate (and optionally,, an, antioxidiUit) is present in ars amount sufficient to mhibit ihefonnadon of an aggregate formal by foe. reaction of a free thiol on a first rtTotechle of ihe protein with a fhse thiol on a second molecule ofthe protein by at least 5-80 % (e:g., at least abaai 5%» at least about 10%, at least about,! $%, at leastabout 20¾. at least about 25%, at least about 305¾. at fesstahoui 35%, ai ieastabout40%, at least about 45%, at least about 50%. at least about.'55%, at least about <8% at. le^f.abaukffS^ii'.at least about/O^u, at least about 75.%., or at least about S0% greater), under jaw-selected conditions, as coarpared to the same composition lacking the carbohydrate (ruid fee antioxidant, if preset). in certain smbodanenis, the: carbohydrate- (and optionally, an antioxidant) is present in am amount sufficient that upon storage, in a gas tight container, at a tefrsperature of 2-8 %>,.fdr a period of 6 isoiiths, the: composition will'retain at least 50, 55,.,60,65, 70,/75, SO, 85, SO, 95,99, or 100 % of the. stability the composition had prior to storage. In a preferred embodiment, the storage occurs in riarkness.

In certain embodiments, the carbohydrate (and optionally, mi antioxidant) is prasentin anamount sufficient,to toe stability comparable to that of a lypphtlized. composition comprising about0.01 % polysorbate-20, pH 6.0,50 mM Citrate, in certain efebofeihenfe, the composition further includes about 1 -40 % (e.g;, about 5 to about 30%,. e.g., about 8 to about 24 %, e.g., about 16 %, e.g., about 3-5 % weight per volume CwTy)} of a carijOh>xirater e. g., sncrosc ior trehalose, fix some embodiments. the carbohydrate is preferably sucrose.

Ih a preferred tanbodimeni, thecompositiotvis a liquid,

In certain embodiments, the composition contains less than about 10 % t>i (e.g,, less than hbOut. 5 % (¾ e g;, le&s-than about 2% 0¾). In a preferred embodimetfe the amount of dissolved O* is less than the amount Ofdissolved inert gases in fee composition.

In certain embodiments, the composition is made by. a method comprising, physical removal ofCfc from fee composiftph (e:g., dogj^slitg/tlfe composition, purging a solution, wife a gas ofeer than 03, e,g., with an. inert gas (e.g,, with N? or At), e.g., bubbling fee gas other than Qj (e.g;,. N; or At) through the eoniposition). la certain embodiments, the protein in the composition that, eowiaim a free thiol has zero, two, four* six, or mors thiol groups which form suithydryl bridges· 6* certain embodiments, ihe piolein amiaining a ftee thiol has two, those, or more tree, feiol groups and has xerfefwo, four, or mole thiol groups which flwin sulfhydryi bridges, per active amt o( protein, Ά in certain embodiments, the .protein epwtaining a.free thiol is seized from the groap cmLSistiftg of gteoeercbri^idase. (G.CB), .basic iibmbi^t §10^ factor (bFGF), .acidic fibroblast growth factor CaPGF); hemoglobin, ihioredokih, calciina* and integnn-binding protein 1 (GIB 1), jb^ta4apftogtebuMn B, beta-laeipgkfemn. AB, serum afbumim antibodies (e.gt, human, antibodies, eg., IgA (e.g„ dimeric IgA), IgG (e.g., JgCS), and IgM; rocombitxant human antibodies), antibody fragments (e;g., Fab* fragmentSi Ffdb'h ftagmentSi finglemhain Fv fragments (scFy)), antibodies arid antibody fragments (e.g., FtA'.ie.gi, mOiioclona:! antibody fragment C46.3; arid sePv) engineered (e.g.y so that the: antibody or antibody fragment eah be kibeled, e.g.,with OpinTc, to climcdt imaging) to introduce cysteine residues (C;g., in die third heavy chain constant domain,. e.g., at position 442 io EU/Ού Humbering; irionoclona.1 miiibody.MN-14 (a hl^-aflmity anti-· carcinoenibfyonic antigen (GBA) m‘ab)); core 2 beta 1,0-N-acetylglueosaminylfa’atisfbfase-M (CIGnT-M), cbm 2 beta tsf>-N-acetyiglucosammyltransferase-I {f3Gnl'-I),pIatejiet.-derived grpwdvfactor receptor-beft (FDOF-b^a), adenine nucleotide tfanslbcase (AbTf ·), p53 tttmOr suppressor protein, giutenprotdds, acid spMngomyelinase(recombinant acid sphyngomyeiinase), desfurpyleefftofur (DFG), apoiij.ioprotein .BrOO (apoB.),and other low density lipoprotein domains^, apoiipoprotein A-i variants (e.g,, apolspoprOtem· A-l (Milano) and appiippprdiem A-I (Paris}), hypoxia-inducible factor-1 alpha (Hlf-I alpha), ?oh Wilibbrand ibetor {VWFj, proteins, and pqitide-mimetics that contain the CAAX motif Ras.)? intveoiydcs, csiboxyjy^tidase Y, cath&psin. B; cathepsin C, skeletal muscle €*?* release ehanneFryanodine receptor (RyRl.)j; unclear ilictor kappa B (NF-KB), AP-1, piotein-disuMde isornsnise (PD1), glycoprotein lb..alphiKGFlb:aipiia),caleme«ri:& (GaN), fibrillin-l, Gf>4, SI(50 A2 (also known as SlOQE), ienoiropic glutamate receptors,, humwintw-alplia-inhibitoi· heavy eivaiii 1, alphiG-antiptesmih (aipiia2AP), thrombospondin (also known as glycoprotein <5)Λ gelsolin, mucins, creatine kinase (e.g.. S-ihiomethylrtnodifred creatine kinase), Factor Vlli, phospholipase B (PLD), insptm receptor beta subunit; acetylcliblmesterage, pmcbyrnoshii modified alpha 2-macixiglpbulm. (alpha 2M) (e.g* proteipass- or ntsShylarnirie-reacted alpha 2M), ghipithionp reductase (OR), complerpent carntipnen;t.C2: (e.g., 2a), complement component C3 (e.g., Cob), eomplensent component 4 (e.g;, 4d), complement Factor B <e.g„Bb)t afeha-lacMbumiti, beta-35-galactosj:da^e,.emiop^mic reticulum. Ct^-ATPase, RHase inhibitor, lipocoftm 1 (also Known as.annexih 1),.proliferating cell nuclear antigen. (PCBA), actin (&g.,.fi6b«kr aotin), tfechzyme A (€oA)> acyi-CoA synthetase (t.g., hmyryl-toetiXyme A syjjlhitoe)/3“2{ra!K-si?oy{-CoA-isQ!tHe^!e precursor, atrial natriuretic feetor (ANF>sansitiVe guanyiate cyclase* Pz-pepiidsse, aldehyde dehydrogenase (e.g., iterated aiddiydedehydrogenase}, P-450 and NADPEHM56 reductase. giyceraidifeydes-3-phosphato dehydrogenase (GAPDB), 0-p>Tuvpyl hdrahydropterin synthetase, iufropin receptor,, low moleculiai weight add phosphatase, Seram choli«<ktorase (BChE), adrenodoXiii., hyaltu«oidase, eanatiiife acyitiMisiBrases, liderteuld«-2 (H.-2), phoaphoglycenUe kinase, insuUnHiegrading efpynie (IDE}, cytochrome cl hrane subunit, S-grotein, vaiy1-bRNAsyMhd^o (VRS), alpha-amylase L rnascie; AMP dsammase, lactate dehydrogenase, arid somatostatin-binding protom; lira preferred embodiment, the protein containing a free thiol is GCB. in another preferred embodiment, the protein containing a free thiol is fcp&F. hr one aspect, the disclosure features h liquid imposition of GCB feat includes GCB, and a carbohydrate at a pH less than 7JO, was produced by exposing the. composition to an in® gas (e.g.,Ήό, said fee inert gas is present in a concctrtrauon higher than in the ambient atmospherei Pig,, the composition cOmdinsat least about 85 90 %, 95 %, or 99 %, or preferably1] 00'% inert gah. In certain cmbodimdilts, the composition also includes an aptfexidant, For example, the antioxidant is cysteine, cysfeitie-HCi, or ntotfemtine |c-g;, present at between about 0,001 and about 10 % (Wfevol)) and the carbohydrate is Sucrose or trehalose (e.g., present at between about i and about 40 % (wt/vol)), in certain embodiments,:th« pH is. in .the range of about 4.5 to about -6,5, o.g., preferably between about 5,0 and about 0.6, e.g.> more prefeasMy between about 5>5 add about 5:8 (e.g., about 5.7). In cortaitoembodimenis, fee coihpositioh alto contains a surfactant {q,g., pploxymer 188).

In one aspect, the disclosure features a composition ihatdneiudes a protein haying a free fesof and a caibohydrattvand is. at a pH below the pKa of a free thiol 6ti fee protein, wherein fee carbohydrate & presen t jp art amount sufficient to increase the stability of the protein at fee pH. Τη certain embodiments, the stabili ty is at least 5-80 % greater {e,g„ at least about. 5%, at least about 1 0%, at least about I 5¾¾ at : least about-20%, at least. about 25%¾ at least about 30%, si leasl about 35%, tit. least about 40%, at legist about 45%, at least about 30%,..at least about 53%, at locust about at least about 65%, at least about.70%, fit least about 75%, or at least about 80% greater), under pre-selected conditions, than the 'stability of a composition which lacks the carbohydrate and which has « pH above the pKa o f a. free thiol, on the protein;

In certain embodiments, the carbohydrate Is present in an amount sufficient to stabilize the free thiol, of thtit protein. ,1a certain embodiments, the. carbohydrate is present ip an amotihi sufficient to inhibit the; reaction of a free thiol on a: first moSecuie of the. protein with a fine thiol on a second molecule of the protein to form an aggregate.

In «stain embodiments» ihe carhphydraie is present in-an amount Mffideat tp inhibit tire: formation of an^aggregate formed by tire reaction of a free, thiol on a first molecule Of the protein with β freOthioi bn a second mdieeaie of the proteiftby at least. 50,55,60,65,71),75,80, 85, 90,95,89, or 100 %, turder pre-selected conditions,as compared fr> the same composition lacking the carbohydrate.

In a preferred embodiment, the carbohydrate is. present m an amount sufficient that upcni storage in da^dess, in a gas ti^t contaiadr, a ttmiparaiure οΓ2<-8°© for a period of up to 3,6,9,12, or 24 months (or longer), the composition will retain at least 50.55,80*65,70,75* SO, S3,90,9s,99,or 100% of ffie.stability it had pdor to storage;

In. certain embodiments, the carbohydrate is presents» anarnouut sufficient for ihe.composi&on to have stability comparable to (bat of a lyophilized cornposj lion, la a preferred embodiment, the composition is a liquid, IncertamembodiraetnSjthecOftrpositioR Contains less than 10 %€>i(e;g., less than 5 % Oh, e,g:, less than 2% Oa). Its certain: endsodiments, tire amount of dissolved O? is less than theamountof dissolved inert gases in the composition;

In certain embodiments, the composition is made by a: method comprising pbysioal rsmoYaf of ©j from ffie composition (s^g., degassing the composition, purspng a solution with a gas other than Oa, e.g., with an inert gasfe..g., with N* or Ar). e;g,, bubbling.die gas other thair <h (e#> Hj or Ar) through the composition),

Jn certain embodiments; the protein ixt the composition, that omiains a. free thiol has two, Uvree, four, five, or more fees thioi groups per active unit of protein.

In certain embodiments, the. protein in' the composition dial contains a. free thiol has two, four, «&, or more thiol groups which, form splfhydryl bribes per active unit (e.g.s dimer) of protein, fit cmam embodiments, the protein that contains a free tliiol has two, three, or more tree tlxro.1 groups and has two, four, or more thioi groups which form sulthydryl bridges, per active unit of protein. ϊη certain embodiments, the protein conmirang a free thioiis Selected front the group coiisistiiig;Of giacoc®?d>r0s3dase tXiOBX.basio fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), he?naglobiri,,thiofedpxiH, calciam- andintegriii-bindtsg protein 1 {OBI), beta-laeioglobulm B,beta-iactoglobulhv AB, serum albumin, amibOdies (e,g., hnaran antibodies, e.g., IgA (e.g., dimeric IgA),. IgG (e.g.,!gCi2), and IgM; reaxmbinaidfiumah anfi^mdies), aitibodyfeagments ( e.g,s Pal/ fragxneotss F(ab’)j fragments, singkrcfrain Pv fragments (seFv)), antibodies and antibody fragments (e.g.-Bab', e.g., monoclonal antibody fimpjdnt C46,3; and scFv) engineered (e.g„ so drat the antibody or antibody fragmen t can be: labeled, e.g,, with 99mTc, to clinical imaging) to introduce cysteine residues (e,.g.,'in the.-third heavy chain constant domain, e.g., at position 442 in EU/OU numbering; monoclonal antibody MN-14 (a high-affinity anti-carcinpembry’-oftiC ahtigeh .(CEA) mab)}, core 2 beta 3,5-N-acetylglucositmm>4txansferaso-M (C2Gn3-M), core 2!heta .1 ,d-N-acetyi.glucosaminyhrajisferaso-1 (C^jnT"%plate3<n-derived growth. teor receptor-beta (PDOF-beta), adenine hhcleotide translochse (ANT), pS3 tumor suppressor protein, gluten proteins, acid ^Wngomyellnase (recombibant acid sphyngomydlinase), d^ furoylcofrioibr (DF’C), apolipoprotein B100 (apoE) and other low density lipoprotein domains, apo lipoprotein A-l variants (o;g* apOIipOprotem ΑΊ (Milano) and spotippprotein A-T. (Paris)), hypoxia-inducibk factor-! alpha alpha), von Wiliebjand ihctor(V'WF), proteins ;and peptide mimetics that cmdainthe CAAX motif (®;g.,)ias),.imtColytics, carboxypepfidaso Y;..cathepsin.B, esathepstn C, skeletal muscle Ca2* reieijse chatmel/ryanodme receptor (f(yRl), hhcleaf factor kappa B (biF-RB), AP-S., prohan-disaUide isomerase (i?Di), glycoproteitt 3 b alpha (GPlb alpha), calcmearin. (Calsi), fibrillin-!, GD4. S100A3 (also, known as SlOOE), ionotropic gSmarnaie receptors. hmuto imfernyiiha^Biabitot^ (afcphj&AJP), tluombospondm (ttfso-lytpWR.38 glycoprotein G), gelsoiin, mucins, creatine kinase (e.g., iS-thiomfeihy'i-modi.iJed creatine Mhase);, Factor Vlil, phospholipase P (PLP), insulin receptor beta subunit, apetylclmljnesterase, prochymosin,. modified alpha 2-raacrogiQbnlju (alpha,2M) (e.g., proteinase- or raethyhmtine-reaefed alpha 2M), glutathione reductase (OR), complement component: €2 (e.g., 2&), complement: component G3 (e.g., C3b), complement component 4 (e.g,, 4dj, complement Factor B {e„:g„ Bh), aipha-lsetalbuinin, beta-il^-gaiachisidasej endOpiasrthc retieulum Ca'^-AXlhuse, R^ase Mubitor, lipoeortin I (also''.knows as annexm 1), prahferitfing cell Unclear imfigen (PCN.A), serin (e..g., globular aetia), e«»uzyme A (CoA), acyI-€oA; synthetase (e.g., fiuiyTyl-coenayme.Asyfltinitase}, 3.-2trans-cnoy1-0>A-tsomerase precursor, atrial nairmretie factor (AFlB)-sensiiive gnariyiate eyciase, Px-peptidase, aldehyde dehydrogenase (e.g.* acyhtted aldehyde dehydrogenase), JMSOanii NADFH-P-4S0 reductase, glyceraldehydes-3-phospltate dtkydrpgenase (GAFDH);, 6-pyruvoyl tetmhyfhoptetin synthetase, luiropih receptor, low nioleculat weight acid phosphatase, serum cholinesterase (RChE), adrenddoxiit, hyshtfomdase, caMtine acyltraasf&ases, interleukin^ (11/-2), phosplioglycsraie fcmaset in$«iii«tegr»ding enzyme f IDE), cytochrome ο I lie?ne subunit, S-protein, YalybtRMA synthetase (VRS), alpha-amylase 1, muscle AMP deanriftase, lactate dehydrogenase, and somatostatin-binding prOtein. in a preferred embodiment, the protein,containing a ftps thiol is GCB.

In another inferred mxbodimeni, the protein containing a, free Urioi ?$ bFGF;.

In One aspect,,the disclosure features a liquid composition of GCB that.includes GCB and a carbohydrate and. is at a pH between about# and about 7,.aari the carbohydrate is present-in an atnount sufficient,to maintain.the biophysical/biochemical integrity, (e.g., molecular weight, charge distribution) atid bioactivitv ciuiraideristies/pTOpetties of the GCB at the pH. For example, die composiritm. retains at least 5Q... 55,60,65,70,75,80,85, 90, .95, .99, pr .100 % of.the jriofogcal aidiviiy it had prior to storage (e.g., storage at a temperature of 2- 8°G for a period of up 10 3,6,9,12 , dr 24 months (of longer)). As another example, at least SO·, 55,60,65, 70» 75, SO, 85,90, 95,99, or 100 % of the proteins'iu the conq>osilion retain the average .molecular weight οι average charge distribution that feepioieinshadprioriosiorage (e.g., ^storage at a temperature of 2-8°€ foraperiod.ofttplo3,6, 9, 12, or 24 months (or longer)).

Id certain; embodbndnte, the pH is i« fife rartgp of about 4.5 to about 6,5, dg., about £.() to about 6.(1 (e.g., feepH is about 5.5 to about 5.8,.e.g.,abont 5..7}.

In a preferred enfemhhusm, thecmbohydrafe is sucrose or trehalose (e..g., present in an amount between about I and about 40 %, e.g., between about 3 % and about 5% (w6vq|)}. in one aspect, fee disclosure features a liquid eoutposition. of GCB that includes. GCBj. ah antioxidant, a eaibohydMe, at a pH between 4,5-6.5, and the composition Was produced by exposing the composition to an inert gas (e,g„ N; or Ar). In. certain embodiments, the pH isin the range of about 4.5 to .about .6,5, e.g.* about 5.0'to about -<yo (e.g., the pH is about 5.5 to about 5.8, e,g.,about 5,7).

In certain fee liquid coniposition indudes about 0.1-40 mg/ml GCB (e.g., more preferably ahoui:Xi.5 to about 10 mgfatl, e.g.f: about :2 to about 8 mgonl or about 5 ni^niti) (e.g., about 2 ntg-hul), about 0.001-10 % cysteine (e.g., about 0.075%), about 1-40 % sucrose (c.g.:, about 16%), at a pH of about 5.5-6.0 (e;g,, about 5;7), and the fevci of dissolved (¾ is less than about 10 % (e.g., less than about 5 %, e:g., less than about 2 %),

In a preferred embodiment, the composition als>6: includes a surfactant (e.g,, poloxamer 188}.

In one aspect* the disclosure features a gas tight container that contains a protein component and a headspace wherein the protein: Component is a protein having a free ihioLajid the headspace is at feast 90%,·95% or 99% (vrri/vpi) an inert gas.

In certain embodiments, fee gas tight container is a prefilled syringe, a vial* or an ampoule. In a more preferred embodiment life prsfilled syringe is a needletess syringe.

Jh certairi.sanbpdiments, the protein containing a free thiol is selected from the group consisUBg of gfecocerebrosidase (GCB), basic fibroblast growth feetpr (bFGfi), acidic fibroblast growth factor {aFGE·},. hemoglobin,, Ibioredoxin, calcium- and MegHn-hliKfing pidtein 1 (CIBl), bte-hfetoglobulm B, be^lactoglobuljn. AB, serum albumin, antibodies (e.g.* human antibodies, e.g., IgA (e.g,, dimeric IgA), igG (e.g.s %G2), and l&Mj recombinant human antibodtes),. antibody iragnients (e,g., Fab* fragments, J?(sb% fragments, smgle^dham 1¾ fragments (scFvJh aniibodies.and antibody fragments (e.g„ Wb't «$, nmnocibnai antUwdy fragment C46.3; and seFv) engineered (e.g., so that the aidiboidy or antibody fragment cat) be labeled, e.g., with WrnXe, to clinical imaging) to Introduce cysteine residues («.g., jn. the third heavy chain constant: domain, feg., & position 442 in EiJ/OU numbering; monoclonal antibody MM-14· (ahigh-aBMty anti-Gar&hdembryonte antigen (CEA) mid»));, core 2 beta 1,6-N-aoOtylglacosatrmiyltrimsfease^M ;(C20nT-M):, core 2 beta 1,6-N-aootylghre^samihylimdsierase-I (C2GrtTH·), platelet-derived growth factor receptor-beta (PDQF'bota), adenine nucleotide hahsioeitse (AMT), p53 tumor suppressor protein, gluten proteins, acid spWngpmyelinase (rewhibinunt acid sphytigomyelinase), d^tbtoy!cefHo&r (DFC), .apohpopTOtein BldO{8|toB) and otherlow density fipopmtem doinaira,; apOirpopmt«in A-I variants (e.g:, apoiipoprotein A-I (Milano) arid apolipqprbttin A- I (parts)), Hypoxia-inducible lacier-1 alpha (HIP-1 alpha), von Willebrand fector (V WP), proteins and peptide mimetics that eontaiu the CAAX motif (e.g i fto),ratK*dydcs> caiboxypeptidase Y, eathepsin B, cateepsin C, Skeletal muscle Ca2, releasechapnej/rytmodine receptor (RyRl)aiueJeSF Stetbr kappa B (NF-1SB), AIM, ptotem-djsuifltle isomemefPBl), glycoprotein lb alpha (GP1 Sr alpha), cateinestrin (CaH), fibrilliw-l, GP4* S1Q0A3 (also known as SlOOE), ionotropicglutamate receptors, hitthtih ihter-rilphp4nhibitor heavy chain l, alpha2-antiplasmin (alphS2AP)i throtnbo&poTidin (also known as glycoprotein G), gelsolin, mucins, creatine kinase (e.g_? S"thiOniethyl-niodmed ct«atine kinase)s Factor V 111. phospholipase D {PLDAirtsUlm receptor beta subunit, acetylehoiinesterase, prochymositi, modified alpha 2-maaOglc&ulin (alpha. 2M) (e.g-, pt&teroase-or methylamine-reastod alpha 2M), idutateionc rodnctase (OR), complement component C2 (e.g., 2a), amtpletnent winponerit G3 (e,g»>C3fc)i.conipiemeftt Component 4 (e.g.v4d), complement Factor B (e,g., Bb), aipha-lactalburain, bem-D^galaelosidase, endoplasmic reiiCulimi Ca^ATFase, RHase inhibitor, hpocotiin 1 (also known as annexin 1), proliferating ceil nuclear antigen (T€NA), aedn (e.g., globular actin), ctasnzyme A(CoA), acyf-CoA synthetase (e.g.* bthyryl-sio^eiiTOe A sjmdi^ase),. S-Strans-ehoyl-GoA^isomerase precursor, atrial nabruretie ihctty (ANiF)-spnairive giiattyHie cyclase, Pz-peptidase, aldehyde .dehydrogenase (e.g.f acylated aldehyde dehydrogenase), P-450.and NADPO-P^Sd pe'ductasc* glyeerhldefedes-3*phbspKMe dehydrogenate (0APDH), 6-pyruvoyl tefehydrppterin .synthetase, lutropin receptor, tow molecuiai weight acid phosphatase, serum. chdlinesti^a8S^BChE,K adrenodoxin, hyaluronidase, erwitine scyltirai}sfemses. interieukm-2· (IL-23, pbosphogi yeerate kinase, insulin-degrading enryme (IDE), cytochrome cl heme.subunit, S-prptdn, uaiyi-t&NAsynthetase (VRS), alpferamyiase l, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein. & a preferred embodiment, the protein containing a free fiwoi is iGCB,

In another preferred embodiment, the protein containing a fee Oiiol is bFGF.

In one. aspect, the disclosure features a methodof packaging acOroposhion that includes: contacting a fee thiol containing protein With an inert gas (e,g., N? Or Ar) to reduce the atnQttni of a reactive species (e.g,, Cb), andinfetiucing the protem.and the inert gas into a gas tigh t oophtihef. 'The. term "‘reactive species” includes &ofee«Ies;or ioris formed by the· incomplete ono*eiectroii reduction of oxygen. These reactive species: include Qsi. snpepoxides; peroxides; hydroxyl radical;, and hypochlorous acid.

In a preferred embodiment, the.Inert, gas is Ah or Ar and the reactive species is (¾. In. certain embodiments., the tree thiol containing protein is selected from the group consisting of glucocefebiosidase (QGB), basic fibmblasigmwih. factor (bFGF), acidic fibroblast growth factor (aFGF), hemoglobin, ihioredoxia, calcium- and integtin-binding ptoleml (CIB1), beia-tacioglobuUn B, beta-laetoglobulin AB, serum albumin, antibodies (c.g;, human antibodies, e,g,, IgA (e>g., dimeric IgA), IgG (e.g.f IgG2),and igMcrecombinajd human antibodies), antibody &a^nentn{ e.g., Bah' fragments, 11(8¾¾ fragments, single-chain Fv fragments fscFv):), antibodies and antibody fragments (e.g., Fab*, e.g., monoclonal antibody fragment C46v3; and seFvr) engineered (e.g,, so that the antibody or antibody fragment caa.be labeled, erg., with WmTc, to clinical imaging) to. introduce cysteiiid residues (e g,, in the third heavy drain constant doruain, e;g., at position 442 in Btl/OU numbering; monoclonal antibodyMN~ 14 (a hif^-affinxiy anti-cdfcihoembryonic pntigsn (CEA) mab)), core2 beta 1 ,·6*·Ν-aeetyi^ncosamiayltfansferase-M (C2GnT-M),core2 beta. acetylglucosanrinyltnmsferase-l {C2GnT-I), platelet-derived gfowtii factor iwebptbr-beta (PfXyF-beta), adenine nucleotide translocase (AKT), gS3 timor suppressor protein, gluten proteins, acid sphingomyelinase (reporabirntm. acid sphyftgontyeiinase}. desinmyleefijofur (DFG), #p3.ipeproiem B3 G0 {ap«B) and other kw density lipoprotein doptains, apoMpopsxjtein A-Ivaritipts (c;&, apolipoprotsin A-ί (Milano) and apolipoprotsih A-3 (Park)), hypoxia-mdueibie factbr-I alpha (HlF-1 alpha),, visa. Willebrand. factor (VWF), proteins and peptide mimetic® that contain the CA&X .motif (evg.^Jias), mucolyties, caiboxypepiidase Y, cathepsin B, eslhqmn C, skeletal muscle Cs1* releuseidmweJ/ryanodine receptor (RyRl), nnckiS· faetor k^ipa B (MF-KB), AP-l, prbteln-diauMide isomerase (PIM)* glycoprotein lb alpha (GPlh alpha), eatcineunn (CaN), fibnlfk-l., Q>4, S1OOA3 (also known. as S3 QOE), ionokopk glutamate receptors, human inter-alpha-inlHhitor heavy chain1, :uiph&2rMrtipi&smiri (aiphaiAPh thrombospondin (also famwn as glycoprotein G), geisoihg mucins, creatine Jdhase (e.g., $^pmp(ii^Me^9^j3«cdia&3(maseK Factor VJIL phosphoiip^e β (FLd), insulin receptor beta subunit, acetylcholinesterase, prochymosin, modified alpha 2-macroglobtiiiu (alpha 2M) (e,g.. proteinase- or methyJamme-reacted alplra 2M), glutatbkme reductase (GR), complement component €2 (e.g., 2a), complement component C3 (e,g., C3h3, complement 'component 4 (e,g., 4dh complement 'Factor 6 (e.g.,Bb), aipha-laciatbumin, beta^-galaetOsidase, endoplasmic reticulum Ca*%ATPase, BNase inhibitor, HpocOitin 1 (also known bs annexin 1), proliferating ceil unclear antigen (PCNA), actin (e;g.,,globular acting coenzyme A (CoA)., acyFCoA synthetase(e.g„ butyrylrooemsyme A. synthetase), S-ltrans-enoybCtoA-isomersse jlrecnrsor, atrial natriuretic factor (ANF)~sehsf!tive guanylate cyclase, :Px-geptidase, aldehyde dehydrogenase (e.g., aeylated aldehyde dehydrogenase),.P-450 and NADPH*fM50 reductase. glycer&ldehydcs-S^ho^jhate dehydrogenase (GAPDR), O-pyravoyl tetr^hydropterin synthetase, Intrepid receptor, low moiecOlai weigh) acid phosphatase, «enun chp1inesteras(.t.(BGhfi)s adronpdoxin, .hyaluronidase, camitnte.aoyltmnsfeni.ses, iaterbalriiv*2 (jfL-2), phosphogfyeeiare kinase, insuttn-degrading mizyme (3DB), cytochrome cl heme subunit, S-protein, vaiyi-tRNA svftth&aseifVRS), alpha~dmyiase I, muscle AMP deaminase, lactate deliydroienase, and spnmto^hn-bmding protetm in a preferred embodiment, the protein containing a free thiol is GCB.

In mother jweferred embodiment, the protein containing a free thiol is bFGih In one aspect, the disclosure features a medtod of treating a patient (e.gv, a patient in ae.edoftm^tm.eni\yith. a-fraa-tiaol containing protein, e.g., apatimi wifha deficiency of the free-thiol protein) that includes administering a composition described herein, e.g., a composition containing a free-thiol protein (e.g., GCB), to a patient. For example, a pharmaceutical composition that is administered to a patient can include a composition described herein, e.g., in a therapeutically-effective amount.

In a preferred embodiment, the administration is by IV infusion or subcutaneous.

In one embodiment, a composition described herein that contains a free-thiol protein (e.g., GCB) is used in therapy.

In one embodiment, a composition described herein that contains a free-thiol protein (e.g., GCB) is used for the manufacture of a medicament for the treatment of a condition in which there is a need for the free-thiol containing protein (e.g., the use of a GCB composition described herein for the treatment of a glucocerebrosidase deficiency, e.g. Gaucher disease). For example, a medicament for administration to a patient can include a composition described herein, e.g. in a therapeutically-effective amount.

In one aspect, the disclosure features a method of treating a patient having a glucocerebrosidase deficiency that includes administering GCB composition described herein.

In certain embodiments, the glucocerebrosidase deficiency is Gaucher disease.

Definitions of the specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a pharmaceutical composition comprising: (i) an antibody or antibody fragment having a free thiol; (ii) a carbohydrate present in an amount sufficient to inhibit aggregation of the antibody or antibody fragment; and (iii) 02 gas, wherein the 02 gas is less than about 10% of the gas in the composition, wherein the pH of the composition is less than 7.0, wherein the composition has less than a 50% increase in an amount of antibody or antibody fragment aggregation as compared to an amount of the antibody or antibody fragment aggregation that was in the composition prior to storage at a temperature of 2-8°C for a period of up to 6 months, wherein the aggregation is measured by size exclusion chromatography, and wherein the composition is made by a method comprising physical removal of 02 from the composition.

According to a second embodiment of the invention, there is provided a gas tight container comprising the pharmaceutical composition of the first embodiment and a headspace, wherein the headspace is at least 90% (vol/vol) of an inert gas.

According to a third embodiment of the invention, there is provided a method of packaging the pharmaceutical composition of the first embodiment, the method comprising contacting the antibody or antibody fragment having a free thiol with an inert gas to reduce the amount of O2, and introducing the antibody or antibody fragment and the inert gas into a gas tight container.

According to a fourth embodiment of the invention, there is provided a method of treating a patient, the method comprising administering the pharmaceutical composition of the first embodiment to the patient.

Other embodiments of the invention as described herein are defined in the following paragraphs: 1. A composition comprising a protein having a free thiol and a carbohydrates wherein the carbohydrate is present in an amount sufficient to maintain the stability of the protein and wherein the pH of the composition is less than 7.0. 2. The composition of paragraph 1, further comprising an antioxidant, wherein the antioxidant and the carbohydrate are present, in amounts sufficient to maintain the stability of the protein and wherein the pH of the composition is less than 7.0. 3. The composition of paragraph 1, further comprising a surfactant. 4. The composition of paragraph 1, wherein the pH of the composition is between about 4.5 and about 6.5. 5. The composition of paragraph 1, wherein the stability is at least 5-80 % greater, under pre-selected conditions, than the stability of a composition which differs by lacking the carbohydrate. 6. The composition of paragraph 1, wherein the carbohydrate is present in an amount sufficient to increase the stability of the protein. 7. The composition of paragraph 1, wherein the carbohydrate is present in an amount sufficient to inhibit the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein to form an aggregate. 8. The composition of paragraph 1, wherein the carbohydrate is present in an amount sufficient to inhibit the formation of an aggregate formed by the reaction of a tree thiol on a first molecule of the protein with a free thiol on a second molecule of the protein by at least 5-80 % under pre-selected conditions, as compared to the same composition lacking the carbohydrate. 9. The composition of paragraph 1, wherein the carbohydrate is present in an amount sufficient that at upon storage, in a gas tight container, at a temperature of 2-8 °C, for a period of 6 months, the composition will retain at least 85% of the stability the composition had prior to storage. 10. The composition of paragraph 9, wherein the storage occurs in darkness. 11. The composition of paragraph 1, wherein the carbohydrate is present in an amount sufficient to have stability comparable to that of a lyophilized composition comprising sucrose, 0.01 % polysorbate-20 pH 6.0, 50 mM Citrate. 12. The composition of paragraph 1, comprising about 1-40 % carbohydrate. 13. The composition of paragraph 1, wherein the carbohydrate is sucrose or trehalose. 14. The composition of paragraph 1, wherein the composition is a liquid. 15. The composition of paragraph 1, wherein the composition contains less than about 10 % o2. 16. The composition of paragraph 1, wherein the composition is made by a method comprising physical removal of 02 from the composition. 17. The composition of paragraph 1, wherein the protein containing a free thiol has two, three, or more free thiol groups and has zero, two, four, or more thiol groups which form sulfhydryl bridges, per active unit of protein. 18. The composition of paragraph 1, wherein the protein having a free thiol is selected from the group consisting of glucocerebrosidase (GCB), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1), beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies, antibody fragments, antibodies and antibody fragments engineered to introduce cysteine residues, core 2 beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta 1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growth factor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT), p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase, desfuroylceftiofur (DFC), apolipoprotein B100 (apoB) and other low density lipoprotein domains, apolipoprotein A-I variants, hypoxia-inducible factor-1 alpha (HIF-1 alpha), von Willebrand factor (VWF), proteins and peptide mimetics that contain the CAAX motif, mucolytics, carboxypeptidase Y, cathepsin B, cathepsin C, skeletal muscle

Ca2+ release channel/ryanodine receptor (RyRl), nuclear factor kappa B (NF-KB), AP-1, protein-disulfide isomerase (PDI), glycoprotein lb alpha (GPlb alpha), calcineurin (CaN), fibrillin-1 CD4, S100A3, ionotropic glutamate receptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin (alpha2AP), thrombospondin, gelsolin, mucins, creatine kinase, Factor VIII, phospholipase D (PLD, insulin receptor beta subunit, acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha 2M), glutathione reductase (GR), complement component C2, complement component C3, complement component 4, complement Factor B, alpha-lactalbumin, beta-D-galactosidase, endoplasmic reticulum Ca2+-ATPase, RNase inhibitor, lipocortin 1, proliferating cell nuclear antigen (PCNA), actin, coenzyme A (CoA), acyl-CoA synthetase, 3-2trans-enoyl-CoA-isomerase precursor, atrial natriuretic factor (ANF)-sensitive guanylate cyclase, Pz-peptide, aldehyde dehydrogenase, P-50, NADPH-P-450 reductase, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyl tetrahydropterin synthetase, lutropin receptor, low moleculat weight acid phosphatase, serum cholinesterase (BChE), adrenodoxin, hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2), phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome cl heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein. 19. The composition of paragraph 18, wherein the protein having a free thiol is GCB. 20. The composition of paragraph 18, wherein the protein having a free thiol is bFGF. 21. A liquid composition of GCB, the composition comprising GCB and a carbohydrate, wherein the pH of the composition is less than 7.0, wherein the composition was produced by exposing the composition to an inert gas, and wherein the inert gas is present in a concentration higher than in the ambient atmosphere. 22. The composition of paragraph 21, further comprising an antioxidant. 23. The composition of paragraph 22, wherein the antioxidant is cysteine, cysteine- HCI, or methionine, and the carbohydrate is sucrose or trehalose. 24. The composition of paragraph 23, wherein the antioxidant is cysteine, cysteine-HCl, or methionine, and is present at between about 0.001 and about 10 % (wt/vol) and the carbohydrate is sucrose or trehalose and is present at between about 1 and about 40 % (wt/vol). 25. The composition of paragraph 21, wherein the pH of the composition is between about 4.5 and about 6.5. 26. The composition of paragraph 21, further comprising a surfactant. 27. The composition of paragraph 26, wherein the surfactant is poloxamer 188. 28. A liquid composition of GCB, the composition comprising GCB and a carbohydrate, wherein the pH of the composition is between about 0 and about 7, and wherein the carbohydrate is present in an amount sufficient to maintain biochemical integrity and bioactivity characteristics of the GCB at the pH. 29. The composition of paragraph 28, wherein the pH is in the range of about 5.0 to about 6.0. 30. The composition of paragraph 28, wherein the carbohydrate is sucrose or trehalose. 31. The composition of paragraph 30, wherein the carbohydrate is sucrose or trehalose and is present at between about 1 and about 40 % (wt/vol). 32. A liquid composition of GCB, comprising GCB, an antioxidant, a carbohydrate, wherein the pH of the composition is between 4.5 - 6.5, and wherein the composition was produced by exposing the composition to an inert gas. 33. The liquid composition of paragraph 32, comprising about 0.1 -40 mg/ml GCB, about 0.001-10 % cysteine, about 1-40 % sucrose, at a pH of about 5.5-6.0, and wherein the level of dissolved O2 is less than about 10 %. 34. The composition of paragraph 32, further comprising a surfactant. 35. The composition of paragraph 34, wherein the surfactant is poloxamer 188. 36. A gas tight, container comprising a protein component and a headspace wherein the protein component is a protein having a free thiol and the headspace is at least 90 % (vol/vol) an inert gas. 37. The gas tight container of paragraph 36, wherein the container is a prefilled syringe, a vial, or ampoule. 38. The gas tight container of paragraph 37, wherein the prefilled syringe is a needleless syringe. 39. The gas tight container of paragraph 38, wherein the protein having a fee thiol is selected from the group consisting of glucocerebrosidase (GCB), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1), beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies, antibody fragments, antibodies and antibody fragments engineered to introduce cysteine residues, core 2 beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta 1,6-N-acetylglucosaminyltransferase-I (C2GnT-l), platelet-derived growth factor receptor-beta (PDGF- beta), adenine nucleotide translocase (ANT), p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase, desfuroylceftiofur (DFC), apolipoprotein B100 (apoB) and other low density lipoprotein domains, apolipoprotein A-I variants, hypoxia-inducible factor-1 alpha (HIF-1 alpha), von Willebrand factor (VWF), proteins and peptide mimetics that contain the CAAX motif, mucolytics, carboxypeptidase Y, cathepsin B, cathepsin C, skeletal muscle Ca2+ release channel/ryanodine receptor (RyRl), nuclear factor kappa B (NF-KB), AP-1 protein-disulfide isomerase (PDI), glycoprotein lb alpha (GPlb alpha), calcineurin (CaN), fibrillin-1, CD4, S100A3, ionotropic glutamate receptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin(alpha2AP), thrombospondin, gelsolin, mucins, creatine kinase, Factor VIII phospholipase D (PLD), insulin receptor beta subunit, acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha 2M), glutathione reductase (GR), complement component C2, complement component C3, complement component 4, complement Factor B, alpha-lactalbumin, beta-D-galactosidase, endoplasmic reticulum Ca -ATPase, RNase inhibitor, lipocortin 1, proliferating cell nuclear antigen (PCNA), actin, coenzyme A (CoA), acyl-CoA, acyl-CoA synthetase, 3-2trans-enoyl-CoA-isomerase precursor, atrial natriuretic factor (ANF)-sensitive guanylate cyclase, Pz-peptidase, aldehyde dehydrogenase, P-450, NADPH-P-450 reductase, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyl tetrahydropterin synthetase, lutropin receptor, low moleculat weight acid phosphatase, serum cholinesterase (BChE), adrenodoxin, hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2), phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome cl heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein. 40. The gas tight container of paragraph 39, wherein the protein having a free thiol is GCB. 41. The gas tight container of paragraph 39, wherein the protein having a free thiol is bFGF. 42. A method of packaging the composition of paragraph 1 , the method comprising contacting the protein having a free thiol with an inert gas to reduce the amount of a reactive species, and introducing the protein and the inert gas into a gas tight container. 43. The method of paragraph 42, wherein the inert gas is N2 or Ar and the reactive species is 02. 44. The method of paragraph 43, wherein the protein having a free thiol is selected from the group consisting of glucocerebrosidase (GCB), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1), beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies, antibody fragments, antibodies and antibody fragments engineered to introduce cysteine residues, core 2 beta 1,6-Nacetylglycosaminyltransferase-M (C2GnT-M), core 2 beta 1,6-N-acetylglucosaminyltransferase-1 (C2GnT-l), platelet-derived growth factor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT), p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase, desfuroylcetfiofur (DFC), apolipoprotein B100 (apoB) and other low density lipoprotein domains, apolipoprotein A-I variants, hypoxia-inducible factor-1 alpha (HIF-1 alpha), von Willebrand factor (VWF), proteins and peptide mimetics that contain the CAAX motif, mucolytics, carboxypeptidase Y, cathepsin B, cathepsin C, skeletal muscle CA2+ release channel/ryanodine receptor (RyRl), nuclear factor kappa B (NF-KB), AP-1, protein-disulfide isomerase (PDI), glycoprotein lb alpha (GPlb alpha), calcineurin (CaN), fibrillin-1, CD4, SI00A3, ionotropic glutamate receptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin (alpha2AP), thrombospondin, gelsolin, mucins, creatine kinase, Factor VII, phospholipase D (PLD), insulin receptor beta subunit, acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha 2M), glutathione reductase (GR), complement component C2, complement component C3, complement component 4, complement Factor B, alpha-lactalbumin, beta-D-galactosidase, endoplasmic reticulum Ca2+-ATPase, RNase inhibitor, lipocortin 1, proliferating cell nuclear antigen (PCNA), actin, coenzyme A (CoA), acyl-CoAsynthetase, 3-2trans-enoyl-CoA-isomerase precursor, atrial natriuretic factor (ANF)-sensitive guanylate cyclase, Pz-peptidase, aldehyde dehydrogenase, P-350, NADPH-P-450 reductase, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyl tetrahydropterin synthetase, lutropin receptor, low moleculat weight phosphatase, serum cholinesterase (BChB), adrenodoxin, hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2), phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome Cl heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein. 45. The method of paragraph 44, wherein the protein having a free thiol is GCB. 46. The method of paragraph 44, wherein the protein having a free thiol is bFGF. 47. A method of treating a patient, the method comprising administering the composition of paragraph 1 to the patient. 48. The method of paragraph 47, wherein the administration is by IV infusion or subcutaneous administration. 49. A method of treating a patient having a glucocerebrosidase deficiency, the method comprising administering the composition of paragraph 21 to the patient. 50. The method of paragraph 49, wherein the glucocerebrosidase deficiency is Gaucher disease.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practicing or testing of the present invention, suitable materials and methods are described below. All cited publications, patent application, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features, objections, and advantages of the invention will be apparent from the description and from the claims.

oetahed DEScRimoN dvcrvitew

Comporitfeps qf tree feiofeqnttontog proteins <s,g., GCB) are relatively unstable m liquid compositions. The three exposed feefeiol groups to CfCB can Undergo reactions which lead to reduction in stability. e.g,. by i%gregafidn of fXlBmqtemles.

For eifenple, in bufferat apHiof 6,typically 1-¾ % o f tbe proteiTi bus aggregated upon one nipnfe of storage and «hoot |·$ &has aggregated after δ ίήόηύτ» .of storage. While not wishing to b&bourtdsfnctly by theoryiormeclvatusm, it is believed that a number of factors contribute to protein instability, e,g,,.tha aggregation reaction: For example, free 0* in solution can accelerate cross-liifetogOf fee fee thiol groups, leading to aggregation. If the reaction foffto fee thiol groups is reduced and/or if the protein can foe made more compact, for example, by burying eystetoe residues in hydrophobic domains, protein aggregation can be reduced. In addition, protean degradation {s.g., fragmentation) cars, be reduced.

Embodiments described herein include one or more measures to address one or more of these issues. As examples, various factors have been addressed to increase the Stability of compositions (e,g., liquidcompositions) of fee fetol-eomaining protems. Cat example: the prcaeitee Of reaefee species (e.g., fee Ob) to Solution, the availability of fee saifhydryl groups (e,g., fee thiols) on the protein, fee proteto conffemrteru and pH. One, two, three, four, or ail of these factors can.be altered or comrolied to Increase the stabi lity of a protein of interest.

Free Thiol-Contai tuna Proteins

Free feioi-bearing proteins are proteins Which to active fbtftr have one or more -gbH moieties, to preferred eriibodtoients, the -feH moiety is accessible to a reactant and can react with feat reactant, e,g., a reducing agent, such as. cysteine, under conditions which are optimal for stability. Alternatively, the -Sr-H moiety can react wife a reactant Under physiological conditions wife one or more biological fluids that it coraes toto contact with when admitnstered to a patient, e-g.,fee moiety is accessible for reaction in. blood.

.V A particularly prefixed frpe ^ol-conffiijiiiug praiein is glaeoeerobrbsidasc ;<GCP). Ths siroettiroof <3€B ia sotuiioa provides relativiefy accessible {as opposed to buried or hindered) free · S-M lnoi^tasrwhkhpFOiftotes reactions with to -#-M moiety.

Anothjgr particularly .prefaced free thtol-coauaning protein is basic fibroblast grop’d» factor (bFGF).

Ctfter«*a®ap|es of free thfed-comahiing proteins include: acidic fibroblast growth factorfaFGF), hemoglobin. dtioi»do^_edliaBJB^ ^;Btognp^fe&ig protein 1 (CIBi), beta^laetoglobuliri B, beta-iactogiobulisa AB, sots albumin, antibodies (e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g., igG2), and IgM; reeombtorit frianan antibodies), antibody fragments ( e.g., Fab* fragments, F(sb’h fragments, singl^oham Fv fegtohtl (scFv)), antibodies and antibody fragments fe.g., Fab*, e.g., monoclonal antibody fragumat £46.-¾ amf soFv) tothavebeea engineered (e.g,, so that to antibody or Mtibotiy fragment 'bait be iabeledi s.g„ witt 99mTcf tor clinical imaging) to introduce cysteine residues (e.g., in to third heavy chain constant domain, e.g., at position 442 in Eli/DU mwtoring; monoclonal antibody MN-14 (a high^affini%r:antotarciftoembryonic antigen (CEA) nisb)}, core.2 beta l,6^N^acetyigiueorimiiny{fra.nsferase-M (C2GoT-M), core 2 beta 1 ;6W^b«^^g!»o0m«»y1fraasF<a^^'I (C:2<jn.T-|), platelet-derived growth factor receptor-beta (PDGFtota), adenine nucleotide tronslooase- {ANT), p53 tumor suppras.ror protein, gluten-proteins, acid s^ngomyeifiiaee' (rooemtHnafttacid sphyfrgomyeitose),, desfm0ylceftiofur;(DFC), apolipoprotein B100 (apoB) and other- low density lipop-oteia domains, apolipoprotein A-i. variants (e^., apoKpoprotein Α-ΐ {Milano) and igiofipoproteiri A-l (Paris)), hypoxia-indltclble factor-1 alpha {HiP-.I alpha), vbri WUfebrand factor (Y'WP), proteins arid peptide numerics that contain tl>e CAAX motif (e.g., Ras)r ;mucolyl'ics, oarhoxypepfrdase Y, eatopsirt B, cathepsin C, skeletal nraecterOr" release channel/ryanodine.receptor {KyR^j itadear factor kappa B (NF-KB), AP-l,prokin-disuliide isomerase (PBl), glycoprotein I b alpha (GPlb alpha), eaicineurin iflaHjvftbTillm- l, CD4, S100A3 (also known as S.100E), ionotropic glutamate receptors, human Inter-alpha-iidiibitorheavy chain i.aipbaZ-anUplasmift (alpba2AP)s thrombospotniitt (also known as glycoprotein (¾ gelsoUn, mucins, creatine kifrasb (e-g., S-driometbyl->mt''jdified creatine kinase). Facto Vilf, .phospholipase D (PLD), insulinrtscepior beta;siibimi1, acetylcholiiiesierase, proehymosin, modified alpha 2-foacrogldlmUn {alpha 2M) (ejg., pmteinase' or tneihylaimne-reacted alpha 2M), g{utatf5i<aier«d»«ase (GR), complement eoraponent €52 (e;g.,2a), complement cotnponeatCS (c.g.. €3b), <»?nplement component 4 {t\g;> 4d),:ex»mpiemeni Factor B (e.g,i B% ^ha4ae® Jbumiii befe-D-galactosidase, cndoplasmie: reticulum Ca2i - ATPaaev iUSfase- inhibitor, lipocortin 1 (also known as annex! κ 1), proliferating cell nuclear antigen (PCHA:)5 actio (e.g.« globular actio), eoentyme A (GoA), acyi-CoA synthetase (e.g., but^yi-eoenzyme A synthetase), 3-2trans-eaK)yl-£'oAriisoiiiverase precursor, atrial natriurdtte factor (ANP)-^siriye:g«aiiytale cyclase, Pz-pepfidase, aldehyde dehydrogenase {e.g.,;acylated aldehyde dehydrogehasch P-450 and T4ADPH-P-450 reductase, giyseraldehydes-S-phospfiate dehydrogenase (GARDIT), 6-pyruvpyl telp^tyrkopterin synthetase, luiropin receptor, low tnoiecaiat height acid phosphatase,; seram cholinesterase (BChE), adrenodoxm, hyalxiroiiidase, camithxe acyliraitsferases, H!terieukii>2 {ll^2),pi^sphoglyfceratttkimiS0i insulin-degrading enzyme (IDE), cytochrome cl herae subumh S-proiein, vajyl-tRNA synthetase (VRS), alpha-amylase I, musefo AMP deaminase, lactate ddiydragenase. and soniaiestatin-bindiftg protein.. Also included are fragments of such proteins (e,g., active domains, structural domains, dominaritjiegaiive fragments, and so forth). The proteins containing a ::free thiol group can be: patently occurring proteins, reconibifumt proteins, proteins modified (e.g.. by recombinant DNA technology) ip contain a cysteine residue, or proteins chenucally cr omzymaficaily treated *P that asulfiiydryi moiety on a cysteine residus is in a reduced State, i.e,,.to have a free*43*H moiety. lit some embodimeatSj a composition described herein includes a protsda having a naturally occurring.s^uoaee, In other embodiments, the sequence of the; protein .will differ at 1., .2,3,4,5, or trp tp. 10 amino acid residues from a naturally occurring sequence, hi mherenfoediments^fiteariiiho acid saiuenec of foe protein will differ by-1, % 3,4,5:, Or up to 10 % train a naturally ptecurring sequence.

Reactive SnefeieS Removal

Re&etivo species, eig., Οχ or.ptirrixides, dissolved in solution, can decrease the. stability of a protein & the composition,, e.g., by proiuoting protein aggregation.

However, evm in the-absence of fife, free -$-H moieties can cross-link. Λ to roemase phftein stabfiit)^ reactive species, eg., Qi in a solution can be removed, e,g<, chemically, by the vise ofQj scavengers,sulfites. Chemical scavengers are often iew-desitableM'lhcy Can cause protein deipadatioii, (¾ can also be •fetuombpiiysicafiy fiftm a solution, e.g., by degassing tbe soiutio», e.g,, by applying a vacuum to the solution to remove the (¾ ftom sotetion and replacing i t withan-iiieat giiS;, e.g., nitrogen or argbru Reduction:of (¾ levels can also be accomplished physically by purging, a solution with a gas other than. Oj, c.g., minert gas, e.g., nitrogen or argon. Purging ejm be accomplished by bubbling the ,:gas through the sola dan to foe purged of

On.

With protefti (e.g^GCB) compositions, bubbling or other tnanipuklions which result 'in interfaces between a gas am! a pmtem-cpntainmg solution mwoften avoided in the treatment of proteins because they can denature proteins, however, these maMpuiatkms have been discovered to be weli-tbierated in the CX7B methods disclosed herein. Οί removahean be combined with minimization of contact Of a solution with ϋ?? e.g.; by manipulation and storage under conditions which minimize the presence ofO& e,g,*> filling of containers under a gas otherthan 0¾ e;g., an inert gas, e.g,, nitrogen or argon, Ortb$:eea!mg of combiners wiftt such a gas. in general, it is: desirable to minimize ite conmet oftheSoUition with Oh prior to administration toiftepaftent Cb levels in head space should be reduced to less than about 10 %, preferably less than abouiS.%, and more preferably Jess than about 2%.

Removal of reactive species may also result in increased protein stability, e;g.,by minimizing pxidhlipn bf other moieliei» as well, «.g^Tyr, Trp, and/or Met residues, in particular* it is desirable to minimize oxidation of these moieties in GOB.

One can test a candidate method for removal of Oj by ptosddmgacon^osiiion contaiitlng 2 mg/trsi GOB, 0:075 % cysteine (its an antioxidant), 16 % sucrose (to decrease ~$-H availability),, adjusting the pH to 5.7, and applying the candidate method. The stability of the;:GCB composition produced by the candidate method, measured, e,g,, as a percent aggregation or degradation, at a predetermined titjife is compared with ode or mote standards. For example, a suitable standard would foe a composition similar to the test condfticn^i except that an Os removal method is not applied. The stabilities, of the treated (Wherein the candidate 0¾ remove!, tfietbod is applied) and unireatedfwhsrein an 02: removal method is not applied) compositions are compared. Suitability can be shown: by the test trfeatmbM.increasing stability as compared With ibis staiidard. Another standard can be' a composi tion similar to the test reposition except Ural in place of the. candidate method .of removal» Oj is removed by a method described herein, for example, by purging or degassing with an inert gas- Suitability eftn be shown by the Candidate method having comparable or better efieets on stability than the method described herein.

Protein stability can bemeasured, e.g,, by measuring protein aggregation or protein degradation. Protein aggregation can.be determineri, e.g., by ske exclusion chremaiogmphy, non-denaturing PAGE, or other methods for determining size, etc. Protein degmlatkin can be determined, e.g., by reverse phase HPLC, non -denaturing PAGE, ion-exchange chromatography, peptide .mapping. Or similar methods.

Antioxidants

The stability of a protein in a composition can be increased, {e;g., ctossrltnkmg .mediated by tree ~S~H moieties can be reduced) by the addition of an antioxidant, and in particular, an miti-oxidant which includes a moiety Which reacts with the free. -.SMi (e.g,, a» -S-H), e.;g., cysteine, cysteine-BCl. or methionine. por a ptoiein (e.g., GOBI that «contains both free thiol groups and also iriremsd disulfide linkage Within the protein molecule, the level of antioxidant (e.g., cysteine) used should be high enough to minmdv^ eresMinltisig of the fee thiol bonds (e.g., aggregation) but low eno.ugh.so as not to cause fragmentation, and/or proteolysis, and/or degradation (e.g., detectable with reverse-phase ΉΡΙΧ2). For example, with cysteine, ikfttctilarjy tor <3CB, inclusion of about. 0.00) % to about 10%, e.g., about Q.Ot to about 0.15 %, e.g., about 0.05 %io about 0.1%, is: suitable. levels over 10 % may not bc-optimai.

For example, one ean test a candidate? antioxidant (winch can be any agent that xian remove or redoesdissolved (¾ ie solution) by providing a composition containing 2 rog/ral GCB, 16 % sucrose (to decrease -S-B availability), adjusting the pH to 5.7, adding the Candidate antioxidant (e,g;, in an. amount described herein, e/g* 0.075%), and jiorpng the composition of (¼. The stability of the GCB composition containing the candidate .antioxidant measured, e.g., as $ percent aggregation or degradation,, at a predeienntocd time is compared with one or more-standards. Fmexampie, a suitable standard would be a cpnjposiliQii: similar to the test conditions exeepttMt an antioxidant is not added to the composition, The stabilities of the treated (containing the ahiioxidant) and,untreated (Inking an antioxidant) compositions are.compared. Suitability can be shown by ihe test treatment increasing stability as compared with this standard. Another standard can. be a composition similar to the test composition except that to place of the Candidate anuoxid&toj a« antioxidant described hereto, for example, cysteine (eg,, to an amount described hereto, e.g„ 0.075%), is added to the composition. Suitahility can he shown by toe .candidate- antioxidant having comparable or better dflhets on stability than aa antfotodahi described hereto. If the candidate mtowpdant is determined to he suitable '(tog,, it increases stability of the composition as: compared to one of the standards), the concehtratton of trie candidate antioxidant can be refined. For example, toe eoncebtraiion can fie increased 0.t decreased over a range of values and compared to the standard and to the other cmicenthdions being tested Jo determine which concentration causes the greatest increase in stability.

Protein stability can be measured,;e.g;, by mensimng.proieiH aggregation or protein degradation. Protein aggregation can be determined, e.g„ bysize exclusion ehromatogratoty, non-denaturing PAGE, or other methods for determtotog Size, etc. toOtein degradation can be detenhined, e.g., by reverse phase HPLC, non-dermtering. PAdE, ion-exchange chromatography, ps^tide mapping or similar methods. A preferred antioxidant is cysteine» Other antioxidants suitable fbr use too! ude-. cysteine-HCL reduced glutathione, toioethanolamine^thiodiglycol, thioacctic acid, monothioglycerol, Nracetyleystetoe, dithiotUreitol, DL-Moetic aeid,.merc3ptoethanoj, dimercaptopropanoi, bisulfite, diliydfoacorbate, metabisulfiteA.suliite, formaldehyde sulfoxylate, thiosulfate, and acetone bisulfite, to some embodiments, acombtoatton of two o.r more of these antioxidants is used to the compositions described hereto. The suitabi lity of the combination can be tested ^ described above for a candidate antioxidant

Addition of mto-oxidaniS can result in increased protein stability% e-g; by mtaumztog oxidationofother xppietjes as \veil, e.g., Tyr, Trp, and/or Met residues. In particular, it is desirable to minimize oxidation of these moieties in GGB.

Carbohydrates imsbrni* embodiments, a fea*i»h^drateis included M foectontjmsifem E;g,, a carbohydMe can cause the protein to be raoip compact,-and for example, bury or. .otherwise hinder access to a moiety, e.g., a.cysteine residue: (e.g,r a free -·&·Β moiety on a cysteine residue), e g., a.eysteme.residue in a hydrophobic domain. This can fe.g,, with GCB) increase protein stability· e;g., by rerlucing prpfein aggregation:.

Carbohydrates ineiude ηοιν-reducing sugars, .&§., non-redticing disacc.harid.es, e.g., sucrose or trehalose, which are suitable for this purpose. The level of sugar i n tire composition can be critical A sugar content of afcdut 1 to about 40 %, e.g., about 5 to gboui 30%, e,g., about $ to ahotC34%,e.g., abouflO %, weight per volume (tv/v) is siiitablej e,g., for .Use with. GC&. A sugar content of about 3 to about S % is also suitable.

One can test ύ «sklidate subsfanee, e.g.,a emididato cafoohydtate, for decreasing •--S-H availability by providing; a composition contforung 2 mgtei GCB, Q.075 % cysteine (as an antioxidant), adjusting the pH to 5.7, adding the candidate substance (e.g., in art «mount described herein, e.g., 16%), and.parging the·^composition of 0*. The stability of Itw :OC0: ipoinin^sUigill xk>t3tajni^ ilse e&idldate st&stahce, measured, e.g,, asa percent aggregation or degradation, at a predetermined' dme is compared with «poor more standards. For example, a suitable standard would be a composition similar to fob test conditions except that a subst ance is nofaddedto the composition. The stabilities of the treated (containing the substance) and untreated (Itu&mg a substance) compositions are compared- Suitability can be shown by the test treahnenimereasmg stability^ compared with fois stfoidard. Another standard ean.be a composition similar to the test composition except that in place of the candidate substance, a substance described herein, for example, sucrose (b.g„ in ah amount described heEdba,·^* 16%)*· is added to the composition. Suitability can be shown by the candidate substance having comparable or better effects on stabi lity than, a substance described herein. I f the candidate .substance is determined to be suitable (e\g., it increases stability of tltocompos&ian as companxi to one of the standards), the concen tration of tire candidate .Tabstfotee cab be refined, Bar example, foe concentration r,ajv be increased, pr decreased oyer a range of values and compared to the^taudard and to the other cpneottoattons being tested to determine which «onceniratiOR causes the greatest increase mutability.

Protein stability can be measured,, e.g,, by measnrtog.preieto aggrpgattop or protein degradation; Protein aggregation ^an be determined, e.g., by size:exchtsi©n chromatography, non-denaturing PAGE, or other methods ibr dstemuning size, etc.

Protein degradation can be determined, e.g.·. by reverse pSmsei lPLC, non-deiMbripg PAGE, iort-exchange chromatography, jseptide mapping, or similar methods.

Preferred carbohydrates: are trehalose dr sucrose. Other preferred substances suitable for use include: maltose; raffmose, glucose, sorbitol. Other Suitable substances that can be used to stabilise the ptoiein include: <»rbohydra«s such as lactose and arabinose; polyols such as matmitol, glycerol, andxylitol; amino acids suchas glycine, arginine, lysine, histidine, alanine, msthioniner.and leucnto: and polymers such as PEG, poloxnraers, dextran, imSyprOpylene glycol, polysaccharides, meihylediulose, sodium catboxymcthyl cellulose, polyvinyl pyrtolidons (PVP), hydrolyzed gelatin, and human albumin. In some embodiments, a combination of two or more of these carbohydrates (e.g., sucrose and trehalose) is used in. the cbmposilions descrilted herein. The suitability of the combination am be tested as described, above for a candidate, carbohydrate;

pH

pH can be critical its achieving an optimized prefeih composition, ,e.g,, a liquid protein composition with inCteased.siability. pH can wtofk by^affecting the eoafdmtatiou and/or aggregation aii&tor degradation and/or the reactiy tty of the protein. For example, at a higher pH, 0¾ can be more reactive; The pH is preferably less than 7.1), more preferably in the range of about 4.5 to about 6.5» more preferably about 5,0 to about 6.0, and more preferably about 5.5 to about 5.8, more preferably abbai 5.7; With some proteins, e.g., GOB, aggregation can reach undesirable levels at..apH above 7.0 and degradation (e.g.,fraginentati0n) c^vreach undesirable, levels at a;pH under 4.5 or 5.0,or at a pH above 6.5 orlfK

Ohs can test a candidate pH by. ptovhUng a composition containing 2 mghr.l G€B, 0.075 % cysteine (as an anriojddant), 16 % sucrose (to decrease -S-H availability), adjusting the composition to a candidate pH, and purging the. composition of 0a, The stability of the GGB composition at the candidate pH, measured, e.g.5 *a» percent aggregation or degradation, at apredetenmned time is compared with one or more standards. For example. asmtablestandard wduUi be a composition similar· to the teSt conditions exeqjt that the pH of die composition is not adjusted. The stabilities of the treated, (the coniposition adjusted to thecandidateipH) and untreated <Qie pH is not adjusted) compositions too compared. Suitability can be shown by the test treatment increasing Stability as compared \vith this standard. .Another standard can be a composition similar to the test compooittoiiexcopt tiurt in place of the candidate pH, the composition has a pH described herein, tbt exteriple, pH 5.7. Suitability ban he shown by tee doftipoaiiiOh.at the candidate pH having comparable or better effects on stability than the Composition: at pH 5.7.

Protein Stability can be measured, e.g., by measm-bg protein aggregation or protein degradation; Protein aggregation csii he determined* e.g., by si» exclusion, .chrorttetogH$hy>,non^enaHirihgPA.GB, or other methods for determining size, etc. Protein degradation can. be determined, eg., by aw&st phase HPLC, non-denaturing PAGE, ion-exchange chromatography, peptide mapping, or similar methods.

Buffers that can be used to adjust the pH of a protein composition include: histidine, citrate, phosphaie^glycine, succinate, acetate, ghhamate, Tds, tartrate, aspartate, maloate, and lactate A preferred buffer is cittate;

Protein Conceriiration A preferred protein GGB) concentration can be between about G. I to about 40 rnghnl, mote preferablyajmaf 0,5 te about tO mg/ml, e.g., abom 2 to about 8 mgdnj w about 5 tng/mi.

Otts can -test for a suitable ptoteiiv ccmcenlration by ptcradihg a composition containing 0.075 % cysteine (as an antioxidant), 16 % sucrose {to decrease --8-4¾ availability), adjusting the pH to 5,7, adjusting the protein .(eg., GCB) to a candidate concentration, and purging the composition of G>. The stability Of the protein (e,g., GC&J composition at the candidate Concenteatioft, measured, e.g., as a percept aggregation or degradation, at a pfedeteraiirted time is compared with one or more standards, For example; a suitable standard would bn a composition similar to the test conditions except that the protein (e.g.„ GCB) concentration is a conctadraifon described herein, e.g., 3 mg/mi, The stabilities of the protein (e,g., GCB) at each concentraiion are coin pared. Soifobtlityoan be shown by the candidate concentration haying; comparable or heifer elects on form a(foncmfoafion described herein.

Frotein stability can be measured, e.g., by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g., by size exclusion ehtorbatographyi npu-denaforihg PAGE, or other methods for determining size, efo.

Protein degradation can be determined,. e g., by reverse phase MPLC, non-denaturing PAGE, ion-exdtaitge chromatography, peptidesnapping, or similar methods.

Surfactants A surfactant can be added to foe liquid protein. {e,g., GCB) composition; In a pidetreii embodiment, this can increase protein stability, e.g., reduce proton degradation, e.g,, due to air/iiquid interface upon shaking/shipment, A surfactant that increases pixttein stifoiliiyve.g., <ioes not came protein degradation, in foe liquid compostdon is selected. A surfactant suitable for use is.e:g., pofoxamcr 18S, e,g., PLURGNIOSS F68.

The surfactant etui. he present in an amount between about 0-005% and aboul,S%,«.g.> between about 0.01% and about 1%, e.g„ about 0.025% and about 0.5%, e.g., about 0.03% and about 0.25%., e.g.* abofit0.04to about 0.1%,d;g;, about 0,95% fo about 0.075%, e.g., 0.05%.

Ideally, a surfactant selected for use in the protein compositions described heroin Is one that is not modified, e.g., cleaved, by foe protein;

For example, od# can testa cmiulidate sutfachs.nt.by providing a cfonpomiion containing 2 mg/mt GOB, 0.075 % cysteine (as an antioxidant), 16 % suerose(tp decrease -S-H availability), adjusting foepH to 5:7¾ adding foeoandidate sudhetarii (e.g·, in an amount described herein, e,g„ .0.;05%), and purging the composition of 1¾. The stability of foe GCB «^nqx^tifot^oQiJtBimng the· candidate surfactant, measured, e.g.,. as a percent aggregation or degradation, at.a predetermined time is compared with one or more standards; For example. a suitable standard would be a composition.sitnilar fo the test conditions except that a $urfacfo&t is not added to the composition. The stabilities of the treated (containing the surfactant) and antreated (lacking a surfactant) compositions are compared in-conditions simulating “real world’’ smiarios, e.gM shipping. Suitability 08« be.shown by the test treatmentihioreasing stability as compared withfeis staiKlard. Another standard can. be a composition. similar to tbe test cen^osition exceptfeat in plan© of theoandidaie-suifaotaitt, a S.urfaotanl4escpb.ed bereitt, ibr example, poloxanter M58 (e;g.,in an amount described herein, e.g„ &b5;%), is added to the composition*

Suitabilty hs; shown by fee candidate surfactant having comparable or belter effects cm stability than a snifactani described herein, if fee cmtdidaie,surfeetiHd is determined to be suitable (e.g„ it increases stability .of fee compo$ition;as compared to one of the sttmfesrds), the concentration of fee canfedatOamfactaot can be tOfmed, For eitamplfe the eonpentratfeean he: increased or decreased overit. rttnge of values and compared to the standard and to the other concentrations being tested to determine which concentration Oausesfhe greatest increase ift stability.

In same enfeodifeentSj a cofebiiuitioapf two dr more surfactfeifs fc bsHSd in fee wmpOSttions described herein. The suitability of the combination can be tested .as. described above for a candidate surfactant.

Protein Stability cap be measured, e.g., by measuring protein aggregation or protein degradation. Protein aggregation can be determined, e.g,, by site exclusion chromatography, non-denatuTing P AGE, or other methods for dcterfiiining size, etc. Protein degradation can be determined, o.g.,,hv reverse pbasb HPLC, nfei-defeduriag PAGE,, ion-exchange chromatography, peptide mapping, or similar methods.

Gaucher disease Is ah autosomal recessi ve lysosomal storage disorder chamctayzed by a deficiency in the lysosomal enzyme, glacocerebrosidasa (GCB). GCB hydrolyzes fee glycb lipid giucoctmebrxVskle that is formed after degradation of glyeosphingolipids in the membranes of white blood cells; and red blood cells. The deficiency in this aizyme,causes glucoccrebroside to accnnndate in large, quantities in the lysosomes ©f plugocytie bells located in fee liver, spleen, and bone marrow of Gaucher patients; Actuntukiion of these molecules «buses a range of elinieai manifestations including splenomegaly, hepatomegaly, skeletal disorder, ferqnfeoqytopenia and anemia. fi&uticsr el at. Gaucher disease; In: 'the Metabolic and Molecular Bases of Inherited Disease (McGraw-Hill, file,· New York* i9.9S)pp.2025-2639);

Tfoahhents for patients suffering from. this disease include administration of analgesics tor relief of bone pain,blood and piatelet transfusions and, in ttotab cases, spleiteotomyi Joint replaeementis sometimes necessary for patients who experience bone erdsiop, Enzyme repkfoefoeni therapy with GCB has been used as a treatmenfribr Gaucher disease;

The structure of GCB in solution provides relatively accessible (as opposed to buried or hindered) fiee--i5-H moieties, which promotes reactions with the ~§-H moiety, GCB can be. obtained by methods that, are known in the art. For example, WC>02/15927, W02005A)89:D47, WQ03/0.S6897, WO01/77307, WO01/07.078, and WQ90/07S73; European Published Ajjp, No. BP1392826; U.S. Published Application Hos, 2003-0026249,.2005-0() 19861,2002-6168750,20O5~0i6508& 2004-004343?, 2.003-()215438, and 2003^1.33924; and U.S. Patent Application No. 10/96%87Θ; GbS. Patent Nos. 7,138,262,6,451,600, 6,074864, 5,879,680,5,549,892,5*2.36,838, ^td 3,910,822 describe methods or preparing GCB protein. Any of the GCB protein preparations described in these patents and applications can be formulated info a. eoritposilion.describefl berein. GCB erhtyniatic activity caa be measured p described in the examples provided herein, or as described in the art., e.g., in U.S. Pat, No, 7*138*262.

Packaging and Delivery

Protein compositions, e.g.;,GCB compositions, e.g., the compositions dpenbed herein and in WG02/i5927, U.S. Published ApplicatioxvNos. 2005-0026249,2005-0019861, and 2002-0168750, apd U.S. Patent Application Noe. 00/641,471 and 10/968,870, can be packaged in a two chamber syringe. For example, the cornpositfon in jyoplufeed form can be placed into a. first syringe chamber and a liquid embe present in. a second syringe chamber (see e.g., U.S. Published Applieafioh No. 2004-0249339).

Pfotein compositions, e.g., GCB cprnppsitkms, e.g., the compositions deserfoed herein and in. WO02/1592?, U,S. Published Application Nos. 2065-0026249,2005-0019861, and 2002-0.168750, and U.S. Patent Application Nos. 09/641,473 and 10/968,870, can fee packaged. m:ft.fteedleles8 syringe {see e,g„ U-S. Patent Nos. &,4fifi,455 and £,919,324). Briefly; t^ one exanipbi, the injectio.n device feclndes: a gas chamber containing a gas or a Mm'be Of gas;; a part Which can allow For Mease of gas from the: gas; chamber;,a ptungcrj which upon the telease of gas from the gas chamber, cm eapse movement of at least, a first piston; a first piston; a second piston; a first chamber, e.g. a-drsmber useful ibi' .drug.stOrage and mixing; a piston housing, in which are disposed the first pistoiij the second piston and the first chamber; a displacement member vybioh sea, independent of the motive power of gas from the gas chamber, cause movement of one or both, of the first, and Second pistons (toe displacement member Can be the plunger or a separate member); an orifice suitable for needleless mjeefiotr in eomnnmication with the fitot chamber; wherein die first and second piston, are sfideably disposed within the . piston housing, md the displacement member, the source of gas, and the plunger are disposed such that: in a first position af.the pistons*, a second chamber, e.g., a fluid reservoir, is defined within the piston housing by the first piston, the piston housing and the second piston, the displacement masher can move one or both of the pistons in to a second position wherein the first piston is in a posifion such that the second chamber, which can be « fluid reservoir, is in communication with the first chamber, which can be a drug storage and mixing chamber, and the second piston is moved in the direction of the first piston, thereby decreasing the volume of tire second chamber arid aBdwifrg five tramrihr of fluid from too second chamber to toe first, chamber, toe plunger, upon release of gas from toe gas chamber, causes toe first piston to move so as to decrease toe volume of the first chamber allowing a substance to be expelled through the orifice add from the chamber and, e.g., tp a subject.

The aeedleless syringe can include separate modules for a fimtcomjtonent, e.g„ a dry or liquid coinpopentyunda 'second oetnj&nenli dig., a liquid component. The modifies cap be provided as two Separate coftqjonsnts and assembled, e.g., by the subject who will admimstcar the component to himself or herself, or by another person, e,g.,hy an individual Who provides or delivers healto: cars. Together, the modules can xonu all or pari of the piston housing of devices described herein. The devices can be used to provide any first and second component where it is desirable to store or provide toe components separately and combine them prior to Mm.inistraiioft.tQ a subject.

Protein (e.g., OCB) compositions described herein can be incorporated mto pharmaceutical compositions suitable ft>r administration to a subject, e.g., a human, A C.jCB composition can include a sufficient dosage of QCB to treat a subject having Gaucher disease, The phaottaceuticai compositions can include one or more pharmaceutically acceptable carriers. As used' herein the language “phiiffiiaceutieglly acc^tabiC earned is inteiuieti to include any arid all solvents, excipients, dispersion mcdtft, coatings, amibactedai and antifungal, agents, isotonic and adsorption delaying agents,and the Like, compatible· with, pharmaccaticaiadmirastraiion. Pbamaceiitical formulation is a welhcslablished art,-,apd is further described, e.g., in German» fed.), Remington:The Science and Practice Of Pharmacy, 20th: ;ed., Lippineott, Williams & Wilkins (20QQJ (ISBN: 06833Cks472); Aiisel et al., Pharmaceutical Dosage Farms mid Drug Delivery Systems; 7th Ed., Lippiiicott Williams &. Wilkins -Publishers (1999} (ISBN: 96'<8330S.727); and Kibbe (e<f), Handbook of Pharmaceutical Excipients American Pharmaceutical Association,·:3κΙ .e& (2000) (ISBN: 09i7330965£j. Except insofar as any conventional media Of agent is mcdmpaiibie' with the active- compound, such media easy he used in the compositions of the invention. Supplementary active compounds cgn also be Incorporated into the compositions, A piiafmaceulical composition may include a “tfeer^seutically effective amount” of a eotoposition described herein. Such effective amounts cap be detomiined based on the effect of the administered eomppsition. A therapeutically effective amount of a composition may also vary acoording to factors such as the disease slate; age, sex„ and weight of the individual, and disability of the composition to elicit a desired response in the individual,, erg., amelioration of at least one symptom of aeo.ndftian.pr disordia:, e,g.. a gjucoccrebrosidase deficiency; e.g:; Gancber disease. A therapeutically-effective amount is sis© one ip which any toxic or detrimental effects of the composition are outweighed by die therapeutically beneficial effects, A. pharmaceutical compositian of the invention is formulated to be. compatible with its intended route of adminisiratibn. For example, the composition can be administered by a: parenteral mode (e,g., intravenous, subcutaneous, intrftperfoopsal, or •ifttram uacuiar 1 eject ion). The phrases “parenteral adminisiruti ©n?i and “administered parenter&Uy” as «sad herein mean modes Of administration other than enteral and topical adfektraiiou, usually by injection, and includes without timitation, intmv<mousx intramuscular, ίηίτ^.ίβηβΙ,.ΙηίΓαΛ^^ irttachpsulaf, intraorbitai, intmcfeae,

Mfeeranal, intrapmtoneal, tnmfecheai, subcutaneous, subcuticular, intinarticute, sabeapsular, subarachnoid, iutraspinai, epidural, and intxnsternaf injection arid infusion. Preferably* the route of administration is intravenous, Solutions of siispensibiis used for parenteral application ban include the Ihiiowing components: a sterile diluent such as: wat^ for injection, saline solation, fixed oils, fplyethyjene glycols, glycerine* pfeylene glycol or other synthetic solvents; antibacterial agents suclv as benzyl alcohol or methyl parpens; antioxidants such as ascorbic acid of sfeUm bisulfite; chelating agents such as ediylenediammetetmacefiC'acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, pH can be adjusted with acids or bases* such as hydrochloric acid or sodium hydroxide; The paietvieral preparation can be enclosed in ampoules, disposable, syringes «r multiple dose vials made of glass or: plastic.

Pharmaceutical compositiOhs suitable for injectable y*e include sterile a^ueoiis shiuiions (where wa:ter.soiubie)or dispersions «md sterilepowders*. e.g.s lyophilked preparations, for the extemporaneous pfepairation o f sterile injectable solutions or dispersion. For intravenous admihislralioti, sui table darners include physiological saline, .bacteriostatic wafer, CREMOEHOE EL™ (BA§F, Farsippany, H.1) or phosphate buffed saline (EBS). in all cases, the txunposition musi .be sterile and should be fluid to the extent that easy syririgabiliiy exkk It.must be stable under the conditions Of manufacture sad storage fed must be preserved against, the contaminating action of nfficmorgautsm s such ashas^fer and fungi. The earrier can be a solvent or dispersion medium contaimng, for example, Water, ethanol* polyol (for example* glycerol, propylene glycol, and liquid polyethylene glvcol, and. the like), ahd suitable niixtures tbereof.. The pntper fluidity can be maiittained, for example, by the use of a coating such as lecithin., by die maininxiance oftherisduired particle size.hi the case of dispersion and by the use of surfactants. Prevention of die action of microorganisms cun be achieved by various antibacterial and antifimgal agents, for example, parabens, eblorobutanol, phenoL ascorbic acid, thiracrosai, and the tike.. In many cases, h will be preferable to include isotonic agents, for examine,, sugars, polyalOOhols stieh as nfetol, sorbitol; sodium cfeMride in the muposition. Prolonged stability of the injectable eomposhidns.can be brought about.by «lauding in theoomposition an agent which, del ays adsavpdorij. for example, aluminum mtinbsfeafate, human serum albmniw and gelatin.

Sterile injectable soteions dfihbfcja-epafed hj^ineorporadng GOB compositions described hcpsm-nt.&e required amount in an appropriate solv<mf witivone or a combination of ingredients «Mftontted abovej::^ requu^ed, followed1 by Shear sterititatian. Generally* dispersions ate prepared by incoqjoratlng &e aefive compound into.* sterile vehicle which contains a basic dispersion meditun and the required otter ingredients from these numerated, above, to the case of sterile powders for toe composition of sterile injectable solutions* the preferred methods of conipositton ate vacuum drying and freeze-drying, e.g., iyophilization, which yields a powder of the active ingredient plus any additional desired ingredient from a previously stmle-fitiefed solution thereof. ft* active comifoiinds (e.g.vGCB compositions described herein) can bo prepared with cmxiisrs that will protect Qte compound against rapid elimination from the body, such as a controlled release fonnuiation, melnding itHplasis and microencapsulated delivery systems. Biodegradable, bfocompatible polymers can bo used, such as ethylene vinyl acetate, palvanhydrides, polyglycolxc acid, collagen, palyarthoesters, and poly-lactic add. Methods for preparation of such formulations.will be apparent to those skilled in die art. The materials cab also be obtained commercially front Alza Corporation arid Nova PhaOTassutieals, toe. Liposomal suspensions (including Upttsomes targeted to infected cells with msatockmtil antibodies to viral antigens) can also be used as pharmitoeutically acceptable carnets. Thesectul be prepared according to methods known to those skilled in the art, for example, d> described In U.S, Pafonf No. 4,522,811..

Protein composilionSi ^g^GCB compositions, 'described herein can be administered with medical, devices known in the art. For example* a protein (e,g.* GCB) composition described herein eM be administered with a needle-Jess hypodermic injection device, such as foe device disclosed.in IfS-Patout N©.:$,399,163,5,383,851, S312325,5,004,413.4,941,880,4,790,824, or 4,596,556. Examples of well-known implants and modules useful 1» the invention include: U:S. Patent Νθί 4>48?γό93, which discloses an implantable mfovo-infosion pump for dispensing medfoatiors at a controlled rate; U.S. Patent No.. 4,486,104, which di scloses a therapeutic device for sdmimstering medicaats through tiie skid; U.S. Patent Ho. 4447,233, which discloses a medication infusion pump far delivering medication at a precise infusion rate; UiS> Patent Ho. 4,447,224, winch disclt>ises ;a \'ariabte .flow in^lantable infusioia apparatus for continuous dmgdeiivery^ O-S. Patent No.,4439,196, which disclose an Osmotic drug delivery system having muiti-chamber compartments; and U.S. Patent No. 4,475,1.96, which discloses an osmotic drag delivery system. Of course, many other Such implants, delivery systems, and modules also are known.

EXAMPLES

Example 1: Materials and Eoainnmrt

The following reagents were used in generating the results presented in Examples2-6: GOB: GlucoeeKhrosidase was,prepared using, but not limited1 to, the methods described m>Meruationaj App, No. PCT/US01/25S82, Other methods known to one of OMipary skill in the art Using recombinant DNA technology may also be used, for example, tire methods described in International App. Nos, P<^/OS8S/043t4,:P<^/Ul^ US.PatisfttlS08. 5,236,83 SB 1, 5,643,670B 1,5,549,89281, and 6,270,989151.

Sitcrose: P/N $-124-01 orS-124-02, Pfausiiehf (Waukegen, 1L)

Cysteine HCJ; P/N 207.1-05, JTBaker (Phillipsburg, NJ)

Poloxainer 188: P/N ΡΠ69, Spectami (New Brunswick. NJ)

Sodium Ctinate: P/N 3649-01, JTBaker (Phiilipsbuig, N.f.) 20 mL vials: P/N 6800-0321, West Pharmaceuticals Services (Lkmvfile, PA) 2 mL vials: P/N 6800-0314, West PhiUmaceutic-4s Services (Lionvills, PA) '20 mm stoppers: P/N 1.950-0414, W9sl Pharmaceuticals »ervic<» (Liotiville, PA) 13 mm stoppers: P/N 1950-0412, West Phamiaceuticals Services (LionviUe, PA) '1% gas: P/N UN1066, Airgas (Salem, ISlIf)

Lyophiliser; Genesis 35EL, SP Industries (Warminster, PA)

Example 2; OCB ΕίοΜΗιν GGB *aw? formulated at 2.5 mg/mL in 16%sucfosei. 0.03% Cysteine HGl, 0.05% pOlo&amef 588,50 mM sodium citrate, pH 6.0; Twenty-ntL glass viais were filled at 4.5 mL, each with die formulated solutions. The filled, vials;were loaded onima shetfof a lyqphilizer and vacuum degassed at 500 mT with, a shelf temperature Of 2G°G for 3 minuteSj followed by backfill with 1% to 950 mBar apd immediately stoppered with 20 nm gray stoppers. The sampJes were plated i»to a 2^eC stability chamber. At % 6,. 12, 18, and 24 months after storage, the samples were pulled and tested for enzyme, activity, aggregation by SE-HPIXI, ami degradation changes by RPddPLC. Enzyme activity was. -assayed by a colorimetric assay using p-nitrophenyl B-D-glueapyiatioside as the substrate (the a'ctmiy cap also be assayed, mg, using die .assay described in U$ Pat, bio, 7,138,262}.

Thexesults arc summarized in Table 1, GCB from this composition had less than 5% changes compared to the baseline· after 24 months at 2-8®C,

Table 1: Stability Sntnosiy for Example 2 after 24 Months Storage a42-8°€

^.Percentage retained from the baseline.

Example 3: Effect of Q? Levels <3CB was.fonnulaiedat 2;5..mgfmL G:CB in 16% sucrose, 0.03% Cysteine HO, 0,05% poloxamer 18%, 50. raM sodium citrate, pH 6.0. Two-wL glass vials were filled to 1 niL each 'with the formulated solutions. The headspace of die vials was treated using a lyOphilizer to have Oi level of 3%, 6%, Or 14%. ihe samples Were placed into a 2-&°C stability chamber, AUbe 6 months time point, fire satisfies were pulled and tested for aggregation change by SE-HPLC and degradation change by RP-BPLC. The results are smtrarijsed in l’abk 2. GCB from these compositions is sensitive, to the oxygen level, in ills headspaceof the vial. With O? Less than 3%, essentMly no changes wem observed after 6 months aiS-S°C.

Tabled: Stabiftty Summary lor Example 3 after 6 Months Storage at 2-8¾

* percentage retained from the.baseline.

Example 4; Effect of Sacruse Levels GCB was formulated at 2.5 mg/mL GCB in .0.05% Cysteine HQ, 0.05% pploxamer 1 88,50 mM sodium citrate, pH 6.0, containing sucrose levels of 0%. 5%* 8%,, or 16%· Two-mL glass vials were, filled tp 1 mi, eadvwaih the fommlated solutions, The vials were vaautfn degased by a lyophitizer arid tiveftaid with ffe to 950 mBar, followed by dosing with 13 pun steppers. The samples were placed into a .2-8¾ stability Chamber. At die 6 month time point, the Samples were pulled for testing of aggregation change by SB-HPLC and degradation change 1?y RP'iiPLC. The results are sunmipiKed in Table-3.

Tallied: Stability Summary for? Exantple 4 after 6 Months Storage at 2-8¾

Percentage retain^ front the baseline.

Example 5; Effect of Ovstelne Levels GC3 was. formulated at 2.5 mg/mLGGB in 16% sucrOset, Q.05% poloxamet 188,. 50 toM sodium citrate, pH 6.0, cotttaimftg cy^teme HGL Of0%.or 0.05%. Two-ral, glass vials were filled to 1 tnL each with the formulated solaticnts. The vials were vacuum degassed by a lyophiUzcf .aftd oveFlaid with N> to 950 niBar in the headspace, followed by dosing with 13 mm stoppers; These samples were placed into a 2-8°C: stability chamber; Anise 6 month ti^iX30J»,The-Stople3i-wafc pulled fox testing of aggi^gaijoii chahge by SE-HPEC «ml degradation change by RP-HPLC. The results are sunimmtred. in Table 4. Addition of cystei.£u; ECh .fed0ce4the aggfeg«ition: tssteJ but ksreasedthe degratiation level as detected by RP-HPLC.

Table 4; Stability Summary for Example 5 after 6 Months Storage at 2-8f‘C

* Percentage retained from the baseliiie.

Example & Effect of nH levels QCft w& jftmnalated at 2.5 mgAaL GCB in 16% sucibse,. ().05% cysteine HC1, .0.05% poioxamcx }$8» 50-mMsodtamvckrate withpH at¢-.0,. 5.8 or. 5.5. TwQ-mL glass vials v/cre filled to 1 m.L .each, with the femjuiated solutions, The viab-were vacuum degassed and Overlaid with'% to f>50ihBar in. the headspace, followed by closing with 15 mm stoppers, These samples were placed Into x 13- (7*C stability chamber. At the 3 -month time point, the samples were pulled for tesftmg of aggregation change by SB· HPiC-an4 degradation change by RP-HP1X. The results am susnmarized in Table 5: Decreasing pH can reduce both the J^^egafioh level and the degradation level.

Table 5: Stability Summary for Example 6 after 3 Months Storage at 13-17C€

* Percemage tstkned Txdm. fbe baseline..

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The term “comprise” and variants of the term such as “comprises” or “comprising” are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Claims

1. A pharmaceutical composition comprising: (i) an antibody or antibody fragment having a free thiol; (ii) a carbohydrate present in an amount sufficient to inhibit aggregation of the antibody or antibody fragment; and (iii) O2 gas, wherein the O2 gas is less than about 10% of the gas in the composition, wherein the pH of the composition is less than 7.0, wherein the composition has less than a 50% increase in an amount of antibody or antibody fragment aggregation as compared to an amount of the antibody or antibody fragment aggregation that was in the composition prior to storage at a temperature of 2-8°C for a period of up to 6 months, wherein the aggregation is measured by size exclusion chromatography, and wherein the composition is made by a method comprising physical removal of O2 from the composition.

2. The pharmaceutical composition of claim 1, wherein the antibody or antibody fragment is a human antibody or antibody fragment.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the antibody or antibody fragment is an IgA, IgG, or IgM antibody or antibody fragment.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the antibody fragment is an Fab fragment, an F(ab’)2 fragment or a single-chain Fv fragment (scFv).

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the antibody or antibody fragment having a free thiol has two, three or more free thiol groups and has zero, two, four or more thiol groups which form sulfhydryl bridges per active unit of protein.

6. The pharmaceutical composition of any one of claims 1 to 5, comprising 1-40 % carbohydrate.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the carbohydrate is sucrose or trehalose.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pH of the composition is between 4.5 and 6.5.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the composition is a liquid.

10. The pharmaceutical composition of any one of claims 1 to 9, further comprising a surfactant.

11. The pharmaceutical composition of any one of claims 1 to 10, further comprising an antioxidant, wherein the antioxidant and the carbohydrate are present in amounts sufficient to inhibit aggregation of the antibody or antibody fragment.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the carbohydrate is present in an amount sufficient to inhibit the reaction of a free thiol on a first molecule of the antibody or antibody fragment with a free thiol on a second molecule of the antibody or antibody fragment to form an aggregate.

13. The pharmaceutical composition of any one of claims 1 to 11, wherein the carbohydrate is present in an amount sufficient to inhibit the formation of an aggregate formed by the reaction of a free thiol on a first molecule of the antibody or antibody fragment with a free thiol on a second molecule of the antibody or antibody fragment by at least 5-80%, under preselected conditions, as compared to the same composition lacking the carbohydrate.

14. The pharmaceutical composition of any one of claims 1 to 11, wherein the carbohydrate is present in an amount sufficient to inhibit degradation of the antibody or antibody fragment, wherein the composition has less than a 50% increase in the amount of antibody or antibody fragment degradation as compared to the amount of antibody or antibody fragment degradation that was in the composition prior to storage at a temperature of 2-8°C for a period of up to 6 months, and wherein the degradation is measured by reverse phase HPLC.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the storage occurs in a gas tight container.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the storage occurs in darkness.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein inhibition of antibody or antibody fragment aggregation is comparable to that of a lyophilized composition comprising sucrose, 0.01% polysorbate-20, pH 6.0, 50 mM Citrate.

18. A gas tight container comprising the pharmaceutical composition of any one of claims 1 to 17 and a headspace, wherein the headspace is at least 90% (vol/vol) of an inert gas.

19. The gas tight container of claim 18, wherein the container is a prefilled syringe, a vial or an ampoule.

20. The gas tight container of claim 19, wherein the prefilled syringe is a needleless syringe.

21. A method of packaging the pharmaceutical composition of any one of claims 1 to 17, the method comprising contacting the antibody or antibody fragment having a free thiol with an inert gas to reduce the amount of O2, and introducing the antibody or antibody fragment and the inert gas into a gas tight container.

22. The method of claim 21, wherein the inert gas is N2 or Ar.

23. A method of treating a patient, the method comprising administering the pharmaceutical composition of any one of claims 1 to 17 to the patient.

24. The method of claim 23, wherein said administering is by IV infusion or subcutaneously. Date: 9 September 2016