CA2136004A1 - Supports having azlactone-functional surfaces, adduct supports, and methods of preparing both - Google Patents

Abstract

2136004 9325594 PCTABS00028 Supports having azlactone-functional surfaces, adduct supports prepared from such azlactone-functional supports, and methods of preparing both are disclosed. Azlactone functionality is introduced to surfaces of a pre-existing support in a manner which retains useful physical and chemical characteristics of the pre-existing support. One method involves exposing surfaces with high energy radiation to generate free radical reaction sites on the surfaces and causing azlactone-functional moieties to react with the free radical reaction sites. Another method involves coating surfaces with azlactone monomers, cross-linking monomers, and optionally co-monomers and polymerizing the monomers to form a polymerized coating of azlactone-functionality on the surfaces. Another method involves dispersion polymerization of azlactone-functional moieties to produce azlactone-functional particles within pores and interstices of a pre-existing support. Adduct supports are formed by coupling nucleophilic reagents, such as biologically active materials, to azlactone-functional moieties of the support.

Description

~ W 0 ~3/25~94 2 1 3 6 0 o i~ PC~r/U~93~0~5~5 SUPPOR~S 8AVINO ~ZLaC~O~E-FUNCT~ON~
SURFACES, ADD~C~ SUPPO~TS, ~ND
~T~ODS OF PREPARIN~ 5aME

Eield of the Invention . Thi9 patent application relate~ to ~upport~ having azlacton~-functional surface~, adduet support~ prepared frem ~uch azlactone-functional supports, and method of preparin~ both.
3_ Azlactone-functional polymeric ~upport~ have be~n prepar~d according to the methods disclo~ed in ~uropean Patent ~ublication 0 392 783 ~Coleman et al.) and ir. European Patent Publication 0 392 ~35 ~Beilmann et al.). In both of the~e publications, example~ show methods of preparation which involve the homopolymeri2ation or copolymerization of azlactone-fun~tionai polymor~ to beco~ the polym~ric ~upport.
Azla~tone-functional moieties are exp~n~ive and valuable. Pr~paration t~chnique- which cau~e azlactone-functional moi~tie~ to be occlud~d from acc~ible use needl~sly wa~tes the valuabl~ azlactone func~ionality.
Al~o, ther~ ia a de~ire to plac~ azlactone-functionality only at ~urfac~c of a ~upport wh~re chemical or phy~ical interaction with other ~ater al~, particularly biologically active matarial~ can occur. Pro~ec~ IV do-cribed in European Patent Publication 0 392-735 disclo~es a method for coating azlac~on2-containing polymer-at - ~
sur~ace~ of sub~trate~. Al~o, European Patent Publication 0 392 735 withi~ the diw lo~ure of ProcRs~ IV and in Example 2~ thereof id~ntLfi~c a de~ise to employ azlactone-containing mono~er3 i~ the-coating proce~s to polymeriz~ the monomer~ in plac~
Thero ar~ a myriad of ~upports which hav~ specific g~ometrio- u~eful for phy~ical interaction with material~, particularly blologically active materials. Th~se support~ have sp~cific phy~ical and chemical characteri~tics: poro~ity, ~urfac~- -area, permeability, solvent re~istance, hydrophilicity, flexibility, mechanical lntegrity, and other ~tability or feature in the u~e ----en~iron~ant, ~tc., which m~t b~ retained for a pro-axi~ting- _ _ support ta r~m~in u~ful. For exa~ple, a microporou~ m~mbrane will not r-main u~d ul a~ a filter if it~ porosity is harmfully compromis~d by the addition of an azlactone-functional moiety to it3 surfacQ~.

SVBSTITUTE SHEES

WO 93/25594 ~ PCr/US93/04~55 213~0~3_1 Monomeric 2-alkenyl-1,3-oxazolin-5-one~ (which compaund~
and homologs thereof are referred to herein a~ 2-alkenyl azlactones and copolymers thereof are known Copoly~ers of 2-alkenyl azlactone~ and olefinically un-aturated monomers and coating~
thereof are disclo-od in U S Pat No. 3 583,950 ~Xollin~ky et al ) Also, copoly~cr~ consl~ting o~ent~ally of a 2-alkenyl azlactone and an acrylic acid o-t-r, and copoly~erization thereof with vinylidene compounds having at l-a~t one hydroxyl group are disclosed in U S
Pat Nos 3,488,327 and 3,598,790 ~oth to Rollin~ky et al ) U S
Pat No 4,695,608 (Engler et al ) di~clo~e~ a bulk polymerization proc~- for free radical polymerization of a vinyl monomer and a monomcric alkenyl azlactono or a macromolecular ~onomer with a molecular weight of los- than about 30,000 in a wiped aurface r-actor such as a twin-screw oxtruder Free radical initiator y-t-m- compri~ing a combination of r~aqents are useul in the proccss Sn~orporat_on of alk nyl azlactono~ into acrylate pr---ure--en~itive dho-ives improv~- th~ adho-ives Al~o di~clo~ed in thi~ patent are m~thod- of preparation of 2-alk~nyl azlactone monomers Summarv of the Inventio~

Thl~ invention provid-- a21actone-functional surfaces on a pre-oxisting support and method- of pr~paring ~uch surface~ in a manner which~retain- u--ful phy~ical and chomical charact~ristic- of i, th- pr---xicting upport Thi- invention al~o provides an adduct support pr-p r-d~fro~ uch azlacton -functional support and m~thods ~ of pr-paring uch adduct -upports _~ The inv-ntion provide- a-che~ically reactiv~ support ' 30 co~pri~inq a pr~-~xi-ting -upport- baving urfac~- and azlactone-functional moietie~ contacting th- uracoc and modifying reactivity of ~uch ~urfac-- wh~le retain~ng u-eful phy-ical and che~ical charact-ri-tic- of th- pro-oxi-ting,-upport ~
~h inv ntion al-o~provid ~ ~ ~ethod of prcparing an azlactone-functional upport, compri-ing-th t-p- of (a) xpo-ing surfac-- of a pr--~xi-ting cupport with high n rgy rad~ation to gen r-te free radical reaction ~te- on th urfac-- and (b) causing szlactonc-functional moi-tl-- to r-act with th- fr radlcal reaction it---to modify ch-mical r-aetivity of th pr---xi-ting ~0 ~upport - _ Tho inv-ntlon al-o provid~--a mothod of pr-paring an azl-cton--functlonal upport, comprl-ing ~a) cov~rLnq ~urfac-- of a pr_ xLzt~ng upport with a21acton~-funct~onal monom-r-, cro-~lLnk~nq monom r-, n~ optionally co-monomer~, and ~b) ~5 copolymerLzing th- monom r- to for~ a cro~-lLnkod, polym~rLz-d, W O 93/25~94 2 1 3 ~ PC~r/US93/04555 azlactone-functional moietie~ at ~urfaces of the pre-ex~ting ~upport to modify chemical reactivity of the pre-exi~ting 3upport The invention also provides an adduct support~
compri~ing a ch~mically reactive ~upport de~cribed above, having S azlactone-funct~onality at surfaces of the ~upport and a ligand comprising ~ nucleophilic reagent reacted wi~h the azlactone-functionality A feature of the pre~ent invention is that method~ of pr-paring th~ azlactone-functional modified surfaco~ do not compro~ise u~eful phy-ical and chemical characteristLc~ of the pre-~xi~ting support Another feature of the pre~ent invention is that azlactone-functlonal moietie- ar~ pre-ent only at urface~ of the pr---xi-tlng ~upport, making efficient use of valuable azlactone-lS functionality Azlactone-functional modified surfaces of a pre-existing support are useful in urface-mediated or catalyzed reactions for synth-si- or ~ite--pecif_c separations Nonlimiting examples of uch u-e- inelude affinity eparation of biomolecules from culture media, diagno-tic ~upports, and nsyme membrane reactors Azlacton--functional modified urface~ are capable of covalently binding azl~c~on~-reactive, nucl-ophilic group~, such as Protein A, - ~ which is a biologlcally acti~ material which re~ersibly binds to an anti~ody, uch-a~ Im~unoglobulin G
2S On method of th- pre-ent invention involve~ the irradiation of surf-ce- of a pre--xi~ting support with high-~nergy radiation to prepar- fre- radical reaction ite~ on guch surfac--upon which azlacton--functional moietLe- can be formed by homopolymeriz~tlon, copolymerization, or gra~ted reaction with free radically roactiv ~21acton--functional moi~tie~
Anoth r method of the pr--ent in~ention involve- the polym~rization or copolymerization of azlactone-functional moieties a~ cro--link d coating- on urface- of pr--exi-ting support3 Another ~Rthod of t~e pre--nt invention involve~ the the ~ --3S di-perzion polymeri2ation of azlacton--functional moi-tie~ to produce cson-lin~ed azlactone-functional particl-- within the pore~
and int-r-tic-- of a pre--xisting porou- upport zlactone~ m-an- oxa201inon moi-ti-- of Formula I - -~0 ~S

WO 93/25594 PC~T/US93~04~
213~i~3`'1 - Rl --N~--R2 ~ t \~ ~ I
o wh~r~i~ R' and R2 independen~ly can b~ an alkyl group having 1 ~o 14 carbon ato~, a cycloalkyl group having 3 to 14 ~arbon atom~, an aryl group having 5 to 12 ring ato~d, an aronyl group haYins 6 to 26 carbon atoms and 0 to 3 S, N, and nonperoxidic 0 hcteroatom~, or R
and ~2 taken together with the carbon to which they arQ joined can form a carbocyclic ring containing 4 to 12 ring atom~, and n i~ an integ~r 0 or 1.
~ Pre-exi~ting ~uppoxt" me~nE a ~atrix having ~urfaces not directly capable of for~ing covalen~ chemical bond~ with nucl~ophilic reagent~, e~peeially biolo~ically activ~ m~t~rial~.
~ Surf~ce~ mean~ both outer ~urface~ of a ~upport ~nd any applicable int~rior surface~ Sorming por~ and inter3tic within a porou~ ~upport.
~ Biologically aetive material~ m~anQ a che~i_al co~vsition ha~ing nucleophilic-funetional group~ and capable of reacting in a manner which affoct~ biological proc~s~.
~ igh energy radiation~ ~an~ radiation of a ~uf f ici~nt doc~ge and energy to cau~e the formation of fre~ radica1 re~sction _ ~iter on ~urface~ o~ upport~. ~igh en~rgy-ra~iation can include elactron-b a~ radiation, gamma radlation, ultraviol~t-(uv) r~ t~on, pla~ma r~di~tion, and corona radiatio~.
It i~ an advantage of ths prQ~ent Ln~ention that snly surface~ o~ a pre-exi~ting support aro ehe~ically-modi~l~dr uch that prsciou~ azl~ctone-functional moi0tie~ are not w~eted within th8 buLk of a matrlx of ~ ~upport being for~ed in the pre~nce of azlactone-functional materi~
It iJ another advantag- of the pre~nt in~ention that sur~aco~ of ~ pr~-exl~ting ~upport as- not phy~ic d ly and chem$c~11y modified in a manner which dimini-he~ bQyond usofuln -a th~ phy~ical and che~ical characteri~tic- for which th~ pre^0xi-ting ~upport waa originally aelected.
~ huD, th~ pre~ent ln~ention retainJ thQ ~ nnfit~ of the phy~cal and ch~mical ch~racteri~t~c~ o~ the ~ulk p~opestlo~ of a pr~-~x~ting ~upport while addlng chem}cal modiflcation of 4S azlactono-functionality to surface~ of a ~upport which re~der~ a W O 93/25594 2 1 3 S O O 1 PC~r/US93/045~

pre-exi~ting support u~eful in waya an unmodified support could not achieve In particular, the presence of azlactone-functional~ty allows for the covalent attackment, without intermediate chemical activation of the ~upport, of nucleophilic-functional-group-containing material~, e~pecially ~iologically active material~
Attachment of ~uch matorials, without intermediate chemical activation of the support, can provide utility a~ adsorbant~, cataly~t~, reagent~, complexing agent~, or purification support~

Embo~ments of the Inven~ion Pre-exi~tin~ Su~orts Selection of a matrix to ~erve as a ~upport can vary widely within the w ope of the invention A ~upport can be porou~
or nonporous, depending on preferred final use ~ ~upport can be continuou~ or non-continuou- depending on ultlmate de~ired usage A
~upport can be made of a vari~ty of material~, including ~upports 20- made of ceramic, gla~-y, metallic, or polymaric material~ or combinations of material~ A ~uppor~ can be flexible or inflexible depending on ultimate de~ired u~age Provi~ion of a~lactone-functionallty on ~urface~ of uch pre-exi ting supports doe~ not adver~ely affect the bulX properties of the pre-exi~ting 3upport, other than providing azlactone-functionality which can react with VariOuJ nucleophilic re~gents without intermediate chemical activation Pr-forred matr$ce- include polymeric supports, ~uch a~
woven and nonwoven web- (such a~ fibrou- wobo), microporcus fiber~, ~
and microporous membrano~ - -Web~
Woven and nonwoven web- are u~eful as ~pport~ having elther regular or irregular phy~ical conflguration~ of 0urface~ from - --which azlaotonQ-funetional moie~ie- can xtend Fibrou~ wobs are particularly de~ired becau~e ~uch web- provide large ~urfaco area~, -with nonwov~n fibrour web- being pr-ferr-d du~ to a~- of manufacturo, low mat-rial co~t, and allowance for Yariation in fiber t~xtur- and fiber d~n~ity A wlde vari~ty o~ f$~er diam-tor-, ~ g , O 05 to 50 micromGt ~, can b- u-~d Web thickn~ can b~ ~arled ~0 wld ly to fLt the application, e g , 0 2 micrometer to 100 om t~ick --or more - -~----r-Fibrou- webs can be prepared by ~ethod~ known in the art, or by ~odlfic~tion~ o~ m-thod- known in th- art Nonwoven web~
can be propared by melt-blowing as i~ known to tho~- -kill~d in the art and di~cloced in, for cxampl-, U S Pat~nt No 3,971,373 In 21~ ~ O G -~ P ~ /Us93/04c~

general, a molten polymeric material L8 extruded in such a way as to produce a ~tream of melt blown polymer mi~ro~iber~ The fiber~ are collee~ed on a collection ~creen, with the microfiber~ for~ing a web Th~ web optionally can be molded or pre0~ed a~ a pres~ure of up to 90 psi to provide an article having a Gurley number of at lea~t 2 ~econds, a~ described in Int~rnational Patent Application Serial Number US 92~07659 The nonwoven web~ can al~o optionally include a permeabl~ oupport fabric laminated to one or both oidea of the web, a~ descri~ed in U S Patent No ~,433,024, or can addLtionally contain reinforcing f~ber- aB de~cribed Ln U S Patent No~
4,681,301 and 4,868,032 The preferred materialH useful to preparB nonwoven fibrou~ web~ include polymer~ ~nd copolymer~ of monomer~ which form fibrous web~ Suitable polymers include polyalkylene~ such as polye~hylene and polypropylene, polyvinyl chloride, polyamide~ such a~ th~ various n~lon~, poly~tyreno-, polyaryl~ulfones, poly(Yinyl alcohol), polybutyl-ne, poly~ethylsne vinyl acetata)~ polyac~ylate~
such a6 ~olymathyl methacrylAte, polycarbon~te, celluloaic- Ruch a~ i cellulo-o acetate butyrate, polye-ter- ~uch ae poly~ethylene terephthalate), polyimide~, and polyurethane~ uch a~ polyether polyurethane~, and co~binations th~rnof Nonwoven web~ can also b- prapared from com~ination~ of 2S co-extruded polymer- ~uch a- polye~ter- and polyalkyl-nes Copolymer~ of th~ monom r- which provid- the above-de-cribed polymer- are al~o includ-d within ths ~COpQ of th~ pr~ent invention Nonwoven web- can al~o be combined web- which are an int~mat- blend of fine fiber~ and crimped taple fiber~
~iber~ and Memb~ç~
Pre-~xi~ting, polymeric support~ can also include microporous ~mbranes, fiber~, hollow^fib~r~, or tubes, all of which aro known ln the ~rt ~he ame material~ u-eful for preparing wob6 ar~ o ~uLtable for prep~rlng f~er~ and membraneJ Pr~~rab1y, membrane~
ar~ compo~ed of homopolymer~ and copolyme~- of polyolefln-Nonlim~ting Xampl-B of uch polyol~fln- ar~ poly-thyl-n-, polypropyl-n-, polybutyl-ne, ~nd copoly~ r~ of thyl-n and ~inyl ~Q acatate -~
A preforred t-chnique us-fu~ for pr~p~r~tion of microposou~ thormopla~tic polymoric ~upport- ~ th~rmally lnduced pha~o ~éparation which involv-s melt blond~ng a thormopla~tic polymer with immL~cible l~quid at a temperatur~ ~ufficient to form. a ~S ho~og~n-ou- mixture, forming an articlQ from the olution into a W093/25594 2l3~nn,l PCl[/US93/04555 desired shape, cooling the shaped article 80 ~ to induce phase s-paration of the liquid and th- polymer and to ultimately solidify~
the polymer, and removLng at least a sub~tantial portion of the liquid leavin~ a microporous polymer matrix Thl~ method and the preferred eomposition~ u~ed in the method are de~cribed in detail in U S Patent No~ 4,95~,943; 4,539,256; and 4,726,989 Alternatively, poly~eric supports can al30 be hydrophobic polyol~fin membranes prepared by thermally induced pha~e ~eparation techn$qu~, but also having a hydrophilic polymeric ~hell interlocked about ~uch hydrophobic membrane surfacQs International Patent Application Serial Number US 91/07686 discloses m~thods of preparation of uch hydrophilized, microporou~ membrane~ using poly(vinyl alcohol) preeursors to form an extrem ly thin poly(vinyl alcohol) shell about the polyolefin urfaeea Alternatively, polymerlo ~upports can be constructed from poly(vinyl alcohol), pr-pared u~ing poly(vinyl alcohol) precursors, to form hydrogel materials, ~uch as di~clo~ed Ln U S
Pat No~ 4,528,325 and 4,618,649 Alternatively, polymeric upport- can be con~tructed from poly(methyl methacrylate) to form other hydrog-l mat~rials Poly~mQthyl methacrylate) i~ commercially avail2bl- and is often u-ed in opthalmic device~ ~uch a~ intraocular l-n-e-, contact len-es, and the like Alternativ-ly, polym~ric gupports can al~o ba prspared by olvent pha~- inver~ion polymerization technique~ Such 2S techniqu-- aro di~clo~-i in U S Pat No 5,006,247 Other SUDDOrtS
C-ramic support~, gla-- upport~, and metallic aupports ar-~all known in~th art ~nd are commercially available or c~n be pr-pared~by a vari ty of known t-chnique- - -_ -Azlactoné-funCtiOnal moie~q~
Azlaetone-functional moiotie- can b- any monomer, propolym r, oligomer, or polymer containing or compri~ing azlactone ---funct$onality of Fosmula I abovc and al-o compri-ing a it~ for fre~ - -35 radlcal reaet~on Preferably, sueh reaetion ~it- i~ a vinylic group-on an un-aturated hydroearbon to whieh azlaeton of Formula I is attaehed Such moi-ti-~ ean bo individual aslactone-eontainLng monom~r-, ollgom r- formed wlth fr-- radical r-actlon lt-- ~nd ha~ing azl~cton--funetionality deriv-d from indi~idual azlaetone-~0 eon~aLn~ng monom r-, or polym-r~ having azlacton--funetionality, - - _- ¦
d~rivod from individual azlaeton--eontaining monomer-, and at least ---on--fre- radieal r-aeting ito Azlactone-eo~t~inine Monom rs ~S Pref-rably, azlaetone-functionality is provid d by 2-WO 93/25~94 ` PCI`/US93/04'~
213~00'1 alkenyl azlactone monomer~ She 2-alk~nyl azlactone monomer~ that can be grafted to or polymerised on surface~ of pre-exi~ting ~upport~ are known compounds, their synthe~is being de~cribed for example in U S Pat No 4,304,705; 5,081,197; and 5,091,489 ~all s Heilmann et al ) Sultable 2-alkenyl azlactone~ include

2-othenyl-1,3-oxazolin-5-one, 2-ethenyl-4-methyl-1,3-oxazolin-5-one, 2-i-opropenyl-1,3-oxazolin-5-one, 2-i~opropenyl-4-methyl-1,3-oxazolln-;-one, 2-~thenyl-4,4-dimethyl-1,3-oxazolin-5-one, 2-i~oprop nyl-4,4-dlmothyl-1,3-oxasolin-5-on~, 2-eth-nyl-4-m~thyl-4-ethyl-1,3-oxa201in-5-one, 2-i-opropeinyl-4-mothyl-4-butyl-1,3-oxazolin-5-one, 2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one, 2-i~opropenyl-4-m thyl-4-dodeeyl-1,3-oxazolin-5-one, 2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one, 2-i-opropenyl-4,4-pentamethylen~-1,3-oxa201in-5-one, 2-i-opropenyl-4,4-t~tram~thyl~in--1,3-oxnzolin-5-one, -20 2--thenyl-4,4-di-thyl-1,3-oxazolin-5-one, 2--thenyl-4-me~hyl-4-nonyI-1,3-oxazolin-5-one, 2-L~oprop~nyl-4-m thyl-4-ph nyl-1,3-oxazolin-5-one, 2-i~opropenyl-4-m thyl-4-~ nzyl-1,3-ox~zolin-5-on~, and ~ 2-ethenyl-4,4-pQntam~thylene-1,3-oxazolin-5-on-, ~h- preferr-d 2-alk nyl azlae~one~ include ~ 2-eth-nyl-4,4-dimethyl-1,3-ox-zolin-5-one (ref-rred to herein a~
- - VDM) and 2-i-opropenyl-4,4-dimethyl-1,3-oxa201in-5-on~ ~ref-rred to h r-in a~ IDM) _ If a eopoly~er io to be for~ed, a co-~onomer having - 30 aimilar or diff-r-nt ch~mical or phy-ic~l properti-- ean b included, dep nding on th de~ired charact-ri~tic~ for the graft or co~ting ~ Nonlimiting xampl-- of eo-monomer- usefuL to b~
copolymRri2-d w$th azlactone-functional moiet~e~-to-form graft- or coatinq- includc~hydroxy thyl m-thaerylat~ ~HEMA~, Yinyl ac-tate, or any of v~nyl aro~atic monom-r-; alpha~ b~ta-un-atur~t~d carkGxylic acids or thoir derivativQ~ or vinyl -t~r~; vinyl alkyl thoro~
olefin~, N-vinyl eompound~; vinyl kotone~ or vinyl aldehydQC
Nonlimiting xumple- of ueh co-~onom r- ar~ di~elo-ed ~n ~urop~an Pat-nt Publ~cation 0 392 735 ~0 Pr-fcrably, HE~A i- u-nd a- a_co-monom r in order to impart hydrophllielty to the azlacton ~unctional urface, in order to faeilitat- eoupling of hydrophilie nucl~ophi~`ic roag-nt~ to for~
adduct support~ -WO 93~25594 2 1 3 ~ ~ O ~ PCI'/U~i93/04~

S~ch azlactone-functional monomer~ can be combined for ~opolymerizing with non-azlactone-functLonal monomers~ in any combination of weight percentage~ to control the reaction resuits For example, u~ing a co-monomer of similar reactivity S ratlo to that of VDM will result in a random copolymer chain grafted to the free radical reaetion ~ite of the ~upport Det~rmination of reactivity ratio~ for copolymerization are dieclo~ed in Odian, Princi~les of PolvmerLzation, 2nd Ed , John Wiley C Son~, p 425-430 ~1981) Alternatively, use of a co-monomer having a higher r~actiYity to that of VDM will re~ult in a block copolymer chain grafted to the reaction ~ite, with little or no azlactone-functional moietie~ near the reactive surface but con~id2rabls azlactone-functionality near the ter~inu~ of the chain Thi- construction lS placs~ azlactone-functional$ty away from sur~ace~ of th~ ~upport (where ~teric hindrance ~ight prcYent th~ coupling of tha azlactone-rcactive nucleophilic reagent)~ but covalently bond~d thereto for considerable r~acti~ity with nucl~ophilic roagents Oli~omers and - PolYmera Although not a~ preferred as ~onomer~, azlactone- ¦
functional prepoly~er- or oligomer- and palymer~ or copolym~rs having at least one frea-radically poly~erizablQ ~ite can al~o be utilizGd for providing azlactone-functionality on ~urface~ of a pre-exi~ting upport 2S Azlactone-functional ol~gomer~ and polymRr~ for ~xample, are prepar~d by fren radical polymesization of azlactone monomerJ
optionally with co-monomer- a~ d--crib~d in U S Pat No- 4,3?8,~ 1 and 4,695,6~ Polym~r~ ha~ing azlactone-functional side chains ~n b~ pr-parec -~ reaetiv- extruAion grafting of azlaetone mOnomBrs to non-azlactone-eontaining poly~ers, u~ing ~UGh techniqu~ a- ~ -di-clo-éd in ~uropean Patent Publication 0 392 783 (Col~man t al ) Nonlimiting example~ of azlactone-functional olig~ers - --and pr-polymer- ~r~ di~clo~ed ~n U S Pat. No-. 4,485,236 and 5,081,197, and Europe-n Pat-nt Publication 0 392 735 In ord~r to ~e u~ful in the pr--ent invontion, th~e azlactonR-~unetional polymer- and prepolymer- mu-t ~e modlfied ~o a~
to al-o compri~- at l-a-t on~ fr~ _ r~dically polym-risabl~ site -~
Thi- i~ r-adily accompli~h-d by reaet~ng a portion of th- azlactone- ~
functional group- with an ~thylenioally un~aturated nuel~ophllic ----compound, ~uch a- those compound~ di-clo-~d in U S Pat No _ _ 4,378,411 ident~ d abov , th-r~by produe~ng a polym r or pr-polym~r having both azlactone-functionality and fre--radically r~acti~- funetionality ~h~ ratio o~ aziactonR moi~tio- to un~aturated m~ieti~ can vary from 99 1 to 1 99, although it i8 W O 93/25594 P ~ /US93/045~
2l3~0ai~

preferable for the azlactone moiety content in the polymer or prepo~ymzr to be at lea~t fifty percent (50~) Method o~ Providing Azlactone-Functional Surfa~e~ on P~e-ex'~ e S Su~20rt8 In general, proces~es for providing the azlactone-functional upports of the pre~ent invention $nvolv- exposing a pre-exi~ting ~upport, especially a pr~--xi~ting, polymeric ~upport, to high energy radiation and to free-radically polymer$zable azlactone-functional moieties Expo~ure of a ~upport to an azlac~one-functional 00$ety can take place either ~ioultaneou~ly with or ~ubs~quent to the irradiation of the ~upport Depend$ng on the the type of radiation and other proce~s cond~t~on~, the a21actone functional polymer which i~ produced can be either ~rafted to the surSace of the pre-exi~ting upport or can be formed as a coating on the support or can becom particles enmeshed within void apace~ of the ~upport In the former in~tance, the azlactone-functional moiety become~ covalently link d to the ~upport, whorea~ in the latter two instance~, it doe~ not R gardless, tAe pre-~xisting support i~ tran-form d into being capabla of forming chemical bond~
w~th nucl-ophilic reagents, e~pecially biologically active matarial~
M~thod- of Trr4~1~tion Pr~-exi~ting upport- aro subj~cted to radiation from a 2S high-en rgy ourc- to for~ fr-o radical ~it~ on or near surfaces of such supports In the ca~- of nonpolymeric 8upport~, no fre~ I
radical ~te~ are formed on urfaces However, during pla-ma or corona treatment, fr-- r-dical ~ite- are formed from th- ~onomer - molec~le~ d-orb-d onto th- nonpolymeric upport su~f ~ce8 . ~igh enorgy ra~iation can b- cla--ified for the pu.r~se~ of th~ pr-~ent invention as either penetratLng or non-penetrating Penetrating radiation i8 utili~ed when on- want- to provido azlactone-functionality to bDth th~ int-rior ~nd exterior surface~ o~-a~pre-exL~ting ~upport, whar-a- non-penetrating r~dia~ion i8 useful to 3S pro~id- azlacton--functionality only to the out~r surface- of the pre-exl~ting ~uppor~
NonlimitLng ex~mple~ of penetrating radiation include b-ta, g~ma, l-ctron-b-am, x-ray, u~ and other eloctromagn-tic radiat~on, wh~r-a- non-penetrating radlation include~ alpha, pla~ma, ~nd corona radiation In oma in~tance-, coron~ rad~ation cun b~
b4co~e pen~trat~ng irradlatlon _ _ P-netratin~ tr~radiation ~ any form- of penetrating radiation are of ~ufficiently high energy, that wh-n ab-orbed by a pr~--xl~ting ~uppo~t, sufficient energy is tran~forred to that 8upport to re~ult in the W O 93~25~94 2 1 ~ ~ O ~ ~1 P~/US93/04555 cleavage of chemical bonds in that ~upport ~omolytic chemic~l bond eleavage re~ults in the formation of a free radical ~ite on the support Thufi, thi~ type of radiation is u~eful when it i~ desired to covalently link the azlactone-funetional moieties, via a free rad~cal graftin~ reaetion, to the ~urfae-s of a pre-~xi~ting ~upport Electron beam and ga~ma radiation are preferrod for thi~
method of grafting du- to the ready-availab~lity of oommercial sourees It ~hould be noted that, although penetrating radiation also generate~ free radical ~ite- within the bulk of many ~upport3, the~ ~ites are genorally not a~ avallablo for reactLon~ with azlaeton--containing moieti-s becau-e such moioties are le~ likeiy to d~ffu~e into the bulk of a ~upport than rcaet at an outer urface of that ~upport Thu-, ven with penQtrating radiation to g~n~rate lS r~act~on sit-s, azlactone-functional~ty i~ u-ually found principally at outsr surfaee~ of a upport Source- of cleetron-bea~ radiation are commercially available, ineluding an En~rgy Scienees Inc Model C~-150 Eleetroeurtain Eleetron Boam Proe~-or Soureo- of uv radiation are 20 ~high and medium prQ-~urc mereury lamp-, douterium lamps, and ~blaeklight emitting 180 nm to 400 nm ~with pref-rred m~ximum inten-ity at about 360 nm) light, which are eomm rcially available f~om a numb~r of vendors, ineluding C n ral Eleetrie Co~pany and GTE
Syl~ania Sourc-- of gamma irradiatLon are comm~rcially avaLlable from~A~omic Enorgy of Canada, Inc u-ing a cobalt-60 high-en rgy ourcs High n-rgy radiation do~agQ-~ar- ~oa-ured in megarad~
(~rad~) or kilogray- ~kGy), whieh i- 1/10 of a;mRad Do-o- can be admini~t-red in ~ingle do-- of th dssir-d lev-l or in multipLe do~ which accumulat~ to th desired l~v~l Do-age- can range cumulativ-ly from about 10 kGy- to about 200 kGy~ and preferably from about 30 kCy- to abou~ 100 kCy- Prof-rably, tho cumulativ~
do~ag- ~xe-ed- 30 ~Gy~ (3 Mrad-) Su~port- ean b- lrradiat d in a packago or eontainor 3S wher~ the t-mp rature, atmoaphero, and oth~r reaetion param tcr- can , b controll-d T~mporature can b- ambient tomp r~ture Th- atmo-ph-r- can b- aLr-or pr-f-rably an in-rt atmo~ph-rs ueh a~ nitrog-n ~
Th~ pr---ur- Ln th- contaLn~r ean be atmo-ph-rie, _ _ ~l-vat-d or d-pr-J~-d to a partial or eompl-t~ vaouum Preforably - =
it i~ at~o-ph-rie D-p-nding upon th- control of th irradlation condition~, upport- can be irradiat-d in a batch or continuou~
- 4~ p~oc~

W O 93~2~594 PC~r~USg3/04~5 213~ G~

Aft~r irradiation and prior to conta~t with the azlactone-func~ional moiety, the atmosph~rQ around the ~urfac~
~hould be kept free of free-radically reactive ~ub~tance~, e~pecially o~
A~ter the fir~t ~top where irradiation forms free radical reaction site~j thc ~cond step provide~ azlactone-functional moietie~ to react with ~uch ~ite~ under ~uitable free radical reaction cond~tion~
Generally, irradiation can take place in the pre~enee or ab~enc- of the azlactone-functional moietie~ Wh~n conducted in the pr--ence of azlactone-functional moietie~, un~raft~d fxee-radical ~co)polymerization can occur in addition to graf ting polymerization A~ a con~-quence, it can be preferred to irradiate a pre-~xi~ting ~upport in the ab~ence of azlactone functional moLetie~ followed by contacting the irradiated upport with azlactone-functionAl ~oietie~ j to initiate ths desired free radical grafting react~on ~hi~ may be accompli~hed by immer~ing the upport in, cozting the ~upport with, or ~praying the ~upport with vapor-, di~per~ion~, or ~olution~
containing azlactone-functional moietiefi- ~lternatively, production of wat~ olublc azlacton~-functionnl polymer~ cdn b~ min~mizsd during irradiation in th- pse-ence of azlactone-functional moisties by incorporation of a multlfunctional cro~-link'ng monom~r Another m~thod of radiation-indueed grafting invol~e~ , irradiation of a polymer film with ionLzing radi~tion in the 2S -pr~¢ne- of ambi~nt oxygen to gen-rat~ hydroperoxide functionality on the surface The peroxide~ are th¢n u~ed to $nitiato graft-polymerizatLon of ol~finic ~onom~r~ by ther~lly induced f r~
radical polym-rization, ccording to t¢chnique~ di~clo~ed in Gupta - et al , Eur Polvm J , 2S (11), 1137 et ~e~ (1989) Alternatively, hydroperoxide ~peci-- can be use~ ~o initLat~ graft polymerizat~on, according to tæchni~ue- di~clo~ed in Yumauchi et al , J A~ ol~m Sci , 43, 1197 et seq (1991) Ultraviolet radiation, which iR a penetrating~-ra~iation - for purpo~e- of th- pre-~nt ~nventioA, i~ differ~nt ~rom other- !
3S pcne~rating radiation~ ln that uv rad~at~on do~- not pro~d- ~nough ~onorgy dir-ctly to st suppor;t- to produce fre~ r~d~caL ~;te~
Th~refore, uv radiation is g-nor~lly conducted ln the pro-enco of - bot~ azlactono-functlonal moi t~e- and photoinitiators, wh~ch sbsorbllght ~n the uv-vi~iblo rang- (250-450 nm) and con~rt thi~ light snergy to chQmical Qnorgy ~n thc form of fr-o radic2~ pecio~
o~n~ration of free r~dical~ by photoinltlator~ q~nor~Liy oc~ur~ by on- o~ two ~roce-~o~, intramolec~lar bond cleavag~ or Lntermol-cular hydrog-n ~b~traction Suit~ble photo~nltlator~ are ldontified in O~t-r t al , ~Photopolymerization of Vlnyl Monom-r~ Chem Re~, 4S 68, 125 (1968) Nonl~niting example~ include acyloln~ and W093~2~594 2l3~nn-l PCI/US93/045~5 derivative~ thereof; diketones; organic Qulf~de~; s acyl dithiocarbamate~; phenone3; ~ulfonyl halide~; and azo compounds. Of the~e possible p~.otoinitiators, azobi~ obutyronitrile), acyloins, ac~loin ether~, and benzil ketal~ and l~phenyl-2-hydroxy-2-methyl-1-propanone (com~ercially available a~ Darocure~M 1173 brandphotoinltiator from E ~erck) are preferred.
The manner in which tho azlactone-func~lonal ~urface i~
imparted to the pre-existing ~upport can ~e nfluenccd by the choice of photoin~tiator. Wherea~ mo~t photoini~iator~ will pro~o-~ ~ree ~adical ~co~polymerization of azlactone-functional moie~ies tO
produc~ coating~, tho~e initiator~ which are prone to abs~raction reaction~, particularly phenones, result in ~imultaneous graft~ng to the pre-exi~ting support. It is pre~erred to utilize cro3~1inking csmonomers witb uv irradiation to minimize the production of ~oluble polymer.
A ~uppGrt can be imm~ssed in, sprayed with, dipped into, or otherwiRe cor.tacted with a mixture, di~persion or solution of azlactone-containing ~onomers, photoinitiator, and op~ionally a cros~linking monomar and/or non-azlactone-containing co-monomer~
Then, the coated aupport i~ expo~ed to uv radiation to cure the monomer~, thu~ re~ulting in the fonmation of azlactone-functional copolymer as a continuou~ or discontinuou~ coating on surface~ of the ~upport.
After rinsing to re0ove unreacted monomer~ and drying, 2S an azlactone-functional ~upport i~ available for nucl~ophilic r-~ction.
Nonl~iting example~ of croa~linking monom~r~ for these azlac~one-functional coating~ includo ethyl0n- qlycol dimethylacrylate (EG~MA), trimrthylolpropane tr~mothacrylate (TMP~NA), methyl~nebi~acrylamide (MBA), and divinylbenzene.
Nonlimiting example~ of co-monomer~ t nclude hydroxyethyl methacrylate ~HEMA), butyl acrylate ~BA), i~ooctyl acrylate (IOA), butyl methacrylato ~BMA), and iso~utyl methacrylate (IBM~).
In ~ome in~tanc~, th~ azlactone-functional cGpolymer i~
3S depo~ited a~ mall partlcle~ or aggr-gato- of ~mall partlclea contactlng the ~urface~ or otherwiJe within the porous tructur~ of th pr~-~xi~tlng ~upport. ~oatlng and uv photopolymerization can occur in ambi-nt conditlon-.
Temperatur~ can be about -~8-C-100C and proferably i8 a~bi-nt.
At~o~pheric condition~ need to b~ lnert uuing non-oxygen ga~ and pr~erably i9 nitrogen or a noble ga~ ~uch as argon.
Alternatl~ely, a w-b coated with tho de~irQd monom~r ~olution can be placed b-twoe~ two oxygen-occlud~ng ~he-t~ that are tran~p~r~nt to 4S tho de-ired typ- of radiatton.

W 0 93/2S5g4 P ~ /US93/045~
213L~On~ :

Since free radical reactione occur quic~ly~ the contact time of the irradiated 8upport with the azlac~one-functional moiety range~ from momentary to le~ than 30 mLn , depending on radiation inten~ity Reaction time~ a~ ~hort a~ a faw 3econd~ are often enough to pro~i~e completed reaction Non-Penetr~tin~ 3~1L~;~e~
Plaema and corona radiation differ from penetratLng irradiation techniques becau~e only the outer hurfac?a of a pre-exi~ting ~upport are ~ubjected to treat~ent with vaporou~ exci~ed azlactone-functional moi~tiee ~hl~ m~thod of irradiation grafting only requ~re~ one ~tep Electrical energy in tAe form of plz~ma dL~charge (al~o knowm a~ glow dlscharge) or corona di~chas~ activat~ the azlaeton--func~ional moietiQ~ in tho vapor ~tato for cont~ct with the out~rmost ~urface~ of the support Tho outer~o~t 3urface~ can includ~ adsorbed monomer molecule~ Without being bound ~o a pasticular theory, it i~ bolieved that th~ oxcit~d azlac~one-functional moieties react with ~urface free radieal ~ite~ leading to the deposition o~ a thin fil~ or network coating the ~uppo~t E~en thouqh ethyl~nically un-~turated mono~er~ ar~ not required for non-penetrating radiation method~ preferably, ~uitable azlactone-functional moietie- are monomoric and covalently react with ~re- radical ~ite~ on th~ ~upport~ ¦
AJ W; th pen-trating irr~iation ~ochniques, one can control the nature of the azlactone-functionality for~od by ~mploying variou~ amount~ of azlactone-iunctional moiet~e~ and non-azlactone-functional moi-tie~ an~ by introducing ~uch amounta into the roaction v~el at difforent t~mee For exa~pl-, one can form a corona-treat~d ~upp~rt ~~
ha~ing a cro~slinked coating or network of VDM ~nd HENA covalently bound thereto Alternatively, on~ can form ~ pla~a-treated ~upport ha~ing layer~ of deposi~ed HEMA and VD~ xtcnding from-th~ ~up~port Alt~rnativaly, onc can tr~at regio-pocific urfacs- of a pr~-existing support 8y pr~venting certain por~ion~ of urfac-~
from being ub~ect~d to corona or pla~a di~chargo tr-at~ont, on~
can produee ~upporto having ~pecific rQgton~ of azlactone^-functionality AlternatlvQly, one can tr-at r-gio~peci~ic urfaces qO o~u-ntially with dif~er~nt azlactona-functional moietle~ t~ produce a complex urSace of a rupport for multiple o~ differentl~in~
nucl-ophilic r~action~
Source~ of pla-~a di~charge energy operate typically at DC, AC, high, r~dio, or microwave fre~uQncieo~ Such ourceo are ~5 comm~rcially available from a nu~ber of vendor~ including ENI ~ow~r W093/25~94 ~13SQnl.~ PCr/US93/045ss Sy~t~ms, Inc. The excitation fsequency is typically a-2 .5 GH2, preferably 2S-125 kHz. The power density at the ~upport~ ~urS ce i8 typically lXl03-0.4 W/cm where the norm~lization i8 b~ed on the projected area of the ~upport (a~ oppo~ed to its actual aurf~ce S area, if porous). Preferably, the power den~ity iB 0.01-0.05 W/cm~.
The gas/vapor compo~ition compri~e~ azlactone-~unctional moieties, either pure os mixed with other organic or inorganic ~apor~ or gace~. Nonl~miting example~ of ~uch vapor~ or ga~ include He, Ar, NC~, C0, 2nd C~; alkane3, alkenes, alkynes; functionalized alkanes, alkene~, and alkyne~; acrylato~, methacrylate~; and other comonomer candidate- identified above with re~pect to copolymerization of azlactone-functional moietie~.
Source~ of corona di~charge energy are available commercially from a number of vendors, including ENI Pow~r Sy~tQm~, Inc. The exci~atlon frequency i~ tyyically S-lO0 XHz, pref~rably 10-50 kHz. The prs~ur~ i- typically 0.5-5 atmo~pA~r~, preferably at or n~r 1 atmo~phere.
~ he power den~ity i~ typically 0.5-6 W~em~, prafer~bly 1-3 W/c~r, when applying the ~ame nor~aLizatlon o~ ~urfac~ ~rea aY
de~cribed with rs-pect to plas~a di-charge above.
Th~ u~ount of depoaitlon of azlactone-~unctiona~
~oietie- can be controlle~ by th- amount of time expos¢d to diccharge. Thc amount of tim- u~ing tha abov- powor den~iti2~ can range from 0.05 ~ec~ to ~ev~xal hours, and preferably fro~ about 1 ~cond to about 5 minute~.
She gas/vapor compo-itlon compri e~ azlactone-functional ~oicties mixed with oth-r org~ic or inorganic ga~-~ or vapor~, from u~ong the candidate- d-~cr~bod above with re-pect ~o pla-ma discharge. Prefor~bly, tho azl~ctono-functional moieti~ have a partial pre~ure sf 1-100 mTorr.
It ha~ recently been pu~ hed in European Pa~ent Publication 0 467 639 (l991) that A pro~o~ b~li6q~d to involve corona di~charg~ can ~ffect~vely achieve ponatrating irradiation effect on nonwovon matRrLal u~ing a ~eliu~ atmocphor~ and diel~ctric protoction ov r both lectrod~ of the corona dl~charge apparatuc.
With thi~ techniguc, ono can e~ploy corona diflcharge of azlactone-functional moieties to render int~rior urface- of a porous upport azlacton--functional. Pow r don~iticc and t~m of d~charqe do~c~ib d above for non-p n-trating irradiation n -d not chango.
4~
Adduc~ Su2sorts and U~e~ulness of tha Inv~ntion Be¢au~- azlactonR-functional moiotie- occupying a ~urfac~ of a pro-exi~ting ~upport ar~ capablo of multipl~ chemical roactiona, azlac~one-WO 93/25~94 PCI'/US93/04~55 213~9~

functional modified ~urface~ of the pr~sent in~ntion can formadduct ~upports Once covalently bonded to or otherwi~e coatin~ a surface, electrophilic azlactone-functional moietie- can react through a nucleophilic ring opening reaction ~t the carbonyl group with any of a myriad of nucl~ophilic reagsnts She result is the formation of an adduct ~upport having pscific reac~i~it$e~
det-rmined by the natur~ of thR nucleophilic reaqent employed in the roaction Nonlimiting example~ of nucleophilic reagent~ LncLude biologically active matorials, acid~, b~-~-, ch~lator~, hydrophile~, lipophile~, hydrophobe~, zwitterion~, detorgsntfl, and any other ch mical whlch can react with the azlactone-function~lity to confer on the surfacs~ of ths pre-exi~ting oupport a modified reactivity lS which differ~ fro~ that which exi~ted on the eupport prior to azlactone-functiqnality modlfication For example, on~ can ~odlfy a hydrophobic surface by reacting on azlactone-functional adduct support with a nucloophilic, hydrophilic moiety Exampls- of nuclcophllic, hydrophilic co~pound- includo poly~Gthylano oxide) comm rcially availabl- a- Je~famins- from S-xaco, Inc ~hu-, uraco- of a ~upport ean becom~ azlacton -funct~onal and then adduct-r-activo, without los~ of the phy-ical and chemical ehAracteri~tic- of uch ~upports such a- poro-ity, flux, color, urface ar-a, p~rmeability, ~olvent re-is~anee, hydrophilic~ty, fl-xibility, m chanical intssrity, and othor ` ~ ctabiLity or featurc in th u-- on~ironm nt Unexpectedly, pre-xi~ting upports can add all of th- b~nafits of azlactone-funct1onality without an effectivQ diminution of the phy-ical and ch-mical charact-ri-tic- of bulk propertie- of the pre-exi-ting ~u~port ~ - --Li~ands and Adduct Su~orts Adduct supportz hav- ligands coupled or otherw~e-tightly bound to azlacton--functional~moieti-~ ext-nding from~
~ùrf~c~- of ~upports to fo biologically or chemically actiqe - ~~ ~`-3S r~action ites For dir~ct coupling, nonl~miting xampl-- of - ,nucl-ophilic lig~nds include primary and ~econdary ine-, alcohol~, and mercaptan~ Of th---, ~mino-functional ligand~ ~r~ e-p-ciaL~y praf-rr d Whilo not being l~mited to ~ p~rticular th-ory, it 1-~-liov d that a ligand form- a covalent bond wh-n coupl-d to_an--azlacton--functLonal moi-ty ~ - _ ~ igand- u-eful for thc prepar~tion of adduct supports can ~1JO vary widoly within tho cope of tho pr-J-nt inv-ntion Pr d -rably, a llgand i~ cho--n ba~-d upon th- cont-mplated end u~e 4S of tho adduct ~upport W O 93/25~94 2 1 3 6 ~ ~ 1 P~/US93/04S~

once ligand3 are coupled to azlactone-functional graft~
or coating~, ~uch ligand3 ar~ available for biological or chemical intaraction, ~ucb a~ ad~orbing, complexing, cataly~i~, or reagent end u~e S Adduct supportQ ar~ u~eful a~ ad~o~bants, complexing agents, cataly~t3, reagents, aa enzyme and other protein-bearing ~uppor~, and a~ chromatographic article~
In a pre~erred a~pect o~ the pre~ent invontion, the ligand d-cired for coupling iB a biologically ac~ive material having azlactone-roactive, nucleophilic-functional group~ Nonlimiting cxample~ of biologically active material~ are sub~Sancea which are biologically, immunoche~ically, phy~iologically, or pharmaceutically act$ve Example~ of biologically active material~ include protein~, peptide~, polypeptide~, antibodies, an~igenic sub~tanca~, ~nzymeq, lS cofactor~, inhibitoss, lectin~, hormone~, s~c~ptos~, coagulation ~actor~ amino acid~, hi~tone-, vitamin~, druq~, cell aurface marker~, and ~ub~tance~ which i~teract with them 0~ the biologicaily ac~ive material~, pro~in~, enzyme~
and antigenic ~ubstance- are desired for ccupling to azlactone-functional support~ Nonlimiting ox~mpl~ of protQlns, enzyme~, and antigenic ubstanc-~ includ- natural an~ recombinant Protein A
~ProtA), Immunogiobulin~ ~uch a~ rat (rIgG), human ~h~gC!, bovin~
~bIgG), rabbit ~rbIgG)~ and mou~e ~mIgG), Concana~alin A ~ConA), ~ Bovinq Serum Albumin ~8SA), Thyroglobulin ~TG), Apo~erritin ~Af), 2S LyJozyme ~y), Carbonic AnhydraJe ~CA), LipaJe, Pig Liver E~tera~e, Penicillin acyla e, and Racterial Antig~n ~8A) U~e~ for immobiliz~d prot~in~, enzymes and antigenic su~tAnce~ are disclo~ed in European Patent Publication 0 392 735 A pre~-ntly prof~rred 4iologically active ~atcrLal i~
ProtA becau~e of it~ multitude of U~B in bio~eparatlon~
Alternat$vely, an adduct ~upport of th~ pr~-~nt invent$on c~n compri~é a coupled ~nzy~e to c~talyse a ch mic~l tran~formation of ~b~tance- recogni2ed b~ th~ enzyme Al~o, a ~upport comprl~ing a coupled antig~nlc ub~t~nco can b- uttlized for affinity purific~tion of a corr~ponding an~ibody from a complax biologLcal fluid flowing through th- porou~ matrix cf the adduct ~upport In other exumples, an ~dduct ~upport having ProteLn ~
coupled to ~nt-rnal and xt-rnal Jurfa~-- can ad-orb bLolo~ic~lly act~v- ma~erial- uch a- Immunoglobulin G for afflnity ep~ration- i proce~-e- In oth-~ example~, an adduct support can b~ u-ed for ~mobilization of antibodio~ or b~ u~d for im~unodiagnosticJ or for W ~orn blotting Alternatively, th- ligand can be a hydrophilo ~or i0proving compatibllity of m-mmalian body implants, such ~8 intraoculsr len~eA, with adjoining tis~u~ On- ~xample of a ligand WO 93/25~94 PCr/US93/045~5 21~C~

especlally 0uit~blo for chemically modifying body implant~ i~ an anticoagulant, such a~ a chemically-modified heparin, e g , an amine-terminated heparin Azlactone-functional moietie~ will undergo nucleophilic attac~ by amine3, thiols, and alcoholn Thu~, lLgands having at lea~t ono amine, thiol, or alcohol group thereon are candidate~ for coupling to azlactone-functional ~urface~ Amine-functional ligand~
are preferred due to ea~e of roaction and ~tability of tha linkage ~o for~ed Coupling of ligand~ to preferred azlactone-~unctional ~urfac~- can u~o method~ of u~ing inorgan~c or organ~c polyanionic salt~ in such concentration~ as to achieve high broad Ypecific biological actiYity for the coupled ligand, auch a~ ~ethods di~clo~od in United State~ Pat No 5,200,471 ~Coleman et al ) lS Coupling of ligand~ to preforred azlac~one-functional surface~ according to the pre~ent invention re~ul~ in adduct fiupports having the for~ula o Rl o wher-in 2S Rl, R-, an~ n aro a0 previou~ly dof~ned, R~ i~ Y or C~, X can be -O-, -S-, -NH-, or -NR' wherein R4 can be alkyl or aryl, and G i- the re~idue of HX~ which perform2 the ad~orbing, complexing, catalyzing, ~eparating, or r~agen unction of the_ adduct support ~
HXG iB a nucleophilic reagent and can be a biologically ~ctivo material f dye, catalyst, reagont, ~nd the like - ~igand- having azlacton--r-active, amine, hydroxy, or thiol nucloophilic functional group- r-act, eith~r ln the pro~enco or ab~enco of ~uitable catalysts, with azlactone-fun~t~onal group~ -by nucleophilic addit~on a- d-picted in the quation _ -wo 93J25~94 2 1 3 ~ PCr/US93/0455~ ~

Rl jN C/,~3~2 O C
O
o Rl o 0 IR~

wh~rein R~, R-, R3, n. X, and G are a~ prQviou~ly d~fined.
Dep~nding on-the func~ional group pr~n~ in the ligand, cataly~t~ may b~ required to achi~ve cff~ctiv~ attsehls~g reaction 20 rat~. Primary amine functional group~ requir~ r~o ca~aly~t~. Acid 1:
c~taly~t~ such aa tri~luo~oac~t~c a¢~.d, cthane~ulfonic tcid, tolu~ne~u~ttonic acid, and the lik~ as~ a~fectiv~ with hyd~oxy snd ~oeond~ry a~ine . unctior~al g~oup~. :
In other a~pectsl of th2 inY~ntion, the ligand i8 not 25 biologic~lly active but ha- other propo~tle~ which lead to it~ ~nd u~o. For ~x~ple, tho ligand ean contain ionic ~unctional groups.
In t~at event, the r~ffultant adducc artiel~ may be util~zed in ~on exch nge ty~e application& Suitsbl~ ~onic gso~ps includ~
ca~boxylic acid, ~ulfonic acid, pho~phonie ~c~d, tertiary a~ine, and 30 quat~rnary amin~ group~ ExampLe~ of u~e~ul ionic group containing ligand~ includ~ aminocarboxyli~, dulfonic, or pho~phonic acid~ ~uch _ a~ ~lycine, alanine, lsueine, Yal~ne, ~-alanine, ~-am~nobutyric acid, 1- and 3-aminopropyl-pho-phon$c aeid, taurine, ~-umLno oct~noio aeid, z~ino~thylpho~phonie aold, amino-metha~ulfonic 3S ac~d, and tho l~ke; hyd~oxy-acld- uch a~ thionic aeid, 3-hy~roxy-propan- ~ulfonic acid, lactic acid, glycolic acid, hydroxyme~hylpho~phonic acid, p-hydroxyb-nzoic acid, nd th~ like~
anA um$no- and hydroxy-funetio~al t~rt~ary and guart-rnary ami~o~
uch a~ 2-di-thyla~inoothylamine, 3-d~r-thyl-aminopropylamino, 40 N,N-~iethyl-thanol-u~ino, and the lik~, and quaterniz-d ~er~ion~
th~rcof Wh n th~ a~ -, hydroxy- or thiol-funct~onal li~and i~ a ~mple al$phatic and/or aromatie hydrocarbon, th~ ro-ultan~ adduct art$cl~ may be u~c~ul in rev~r~ pha~Q or hydrophobic interaet~on typ~ chromatographic proc~--o~ R~actlon of thQ ~upport of thi~
45 invontion with ~ery hydrophilLc or hy~rophobie li~and~ c~n b- u~cd WO 93/25594 PCI/US93/04~

213~~ ~-to produce adduct articles di~playing highly ab~orbant propertles toward~ aqueous or oily fluids, re~pectively Other type~ of ligand~ and use~ will be obviou3 to one skilled in the art and are conQidered to bQ within the ~cope of the pre~ent invention Object~ and advantage~ of thi~ invention are further illustrated by the following example~, but the particular matsrial~
and mount~ thereof recited in the~o x~mples, a~ well a~ other condition~ and d~tails, ~hould not be con~trued to unduly limit thi~
invention Examole~
~mple 1 - Preirradiation Electron 8eam Gr~ftinc of ~dro~hobic PolYethvlene ~P~) Nicro~orou~ Mem~brane with 2-VinYl 4 4-dimethvla21actone lVDM~
A PE microporous membrana, prepared ac~ording to tbe method of Example 23 of U 5 Pat No 4,539,256 (Ship~an), having a pore ize of 0 496 ~m, a thickne~s of 73 9 ~m and a void volume of 81 '~, wa~ passed through an electron ~eam (e-beam) chamber within a ~odel 1 Elcctrocurtain C8-300l30/380 (manufactured by Energy Sc$ nce-, Inc , Wilmington, MA) to gen rate free radical- on the m mbrane Th acc-ler~tinq voltag- of the e-~cum wa~ 150 KV, with total irradiation do-- of 50 kGy- ~5 Nrads) ~embrano sample~ (7 6 X 12 7 cm) wer- pa-~-d through th- -b am equipm nt taped to a poly-~ter carrier web travelinq at 6 L m/min 2~ The ampl-- xited th -b am chamber directly into a N, purg d box, wherr they w re removed from the carrier and immer-ed into a olution of VDM ~SNPE, Princ-ton, NJ) di~-olved in thyl ac-tate She inert atmo-pher- in th- glove-box wa~ intended to pr-v-nt~proma*ure qu~nching of the gen rated radical- by oxygen The~onomer -olut on- had cone-ntration- of 25, 50, and 10Q volume-pereent VDM and had b-~n purged with argon for 1 h to di-plac~ any dL--olv-d oxyg n Irradiated m~mbrane~ were -oaked in the monomer ~olution for 3 to 5 min followed by~a 5 min ~oak in pure thyl- ~
ac-tato to wa~h out ~xe~zz monom-r They were dried and placod in 3S zip-lock bag- to prevent pos-ibl- hydr~ly~i~ of the azlacton- by atmocpheric mo$~ture Four$er-tran~form Lnfrared p ctro~eopy ~FT-T~) wa- u~ed- --to eharacterizo the grafted m mbr-n-- The ratLo of th- azlactone carbonyl ~b-orptLon ~1824 cm-l) to th PE C-H band ~1462 cm-l) givè~
40 , a relatLv- m ~-ur- of th- bound azl~ctone ~atio- of 0 023, 1 78t and 1 27 w~r~ found for th ~umpl-~ r-act-~ with 25, 50, and 100 VDM, re-pectively So confirm that ~11 of,th- VDM wa~ inde-d covalently gra~tcd to th- mombran-, the ~ample- wer- xt~acted by thr e 15 min 4S ~oaking~ in pur- thyl ac-tate ~thre- replicate~ of ach) Weight W093/25594 ~l350nl~ PCr/US93/045 los~ ~alues were 1.1, 0.7, and 0.0~. Since there wa~ a weight gain of at leaQt 10~ during the graftin~ step, it wa~ concluded that 90 or more of the VD~ wa~ covalently bonded.
.
ComParisOn Exam~le 2 - Mutual Irradiation ~ eY~ Graftin~ onto PE
Micro~orous Membrane~
A~ a demonstration of the advantage of preirradiation graftsng over that of mutual irradiation, the 3ame microporou~ PE
membrane a3 used in Fxample 1 wa~ saturated with 10~ (w~v) VDM/ethyl acetate ~olution ju~t prior to being pa~ed through ths e-beam chamber for expo~ur~ a~ a dose of about 50 ~Gy. The mQmbrane w~
rin~ed in pure ethyl acetate for about S min. upon emerg~nce from the e-bQam chamber to remove unpolymerized monomer. The azlactone:PE ratlo by FT-IR wa~ 1.39, indicating that a substantial lS amount of the YD~ wa~ indeed ~ound to the meFbrane. The memb~ane weight decreased by 10~ following the extended 801~ent ~toaking procedure descr_bed ln Exa~ple 1, indicating that a ~ignificant portion of the ~ wa~ not ~rafted to 'ha membrnne. It i~ al~o---likely that homopolymor~ of VD~ (not bound to th~ m~mbrane~
aceounted for much of the VDM which wa~ not r~adily wa~hod from the membrane becaus~ of ~ntrap~nt within the mem~rane.

Exam~le_3 - ~reirradiation E-beam Graftina of Hvd~ophLl~zed P~
~icro~orous ~e~Lbra~e~ i Tho starting PE~ microporous membran~ f rom Example 1 wa~
hydrophil~zed by coating the internal and cxternal po~e qurfaces w~th a 4~ (wlv) ~olution of poly~vinyl trifluoroac~tat~) ~PVTFA) f ollow~d by reaction with ~onia ga- to convert th~ PV7~FA to highly cryctallln~ poly(vinyl alcohol), a hydrophilic polym~r, u~ing the proc~dura~ d~cribed Ln Exa~pl~ 7 of coa~igned, cop~n~is~g Unit~d State~ Patent Application Ser. No. 01/775,969 ~Gagnon et al.) = publi~hed ~- PCr Publication W0 92/07899 ) . Cra~ing cond$tion~ were~- the ~ame a3 de~cri~ed in ~x~mple 1 except for tho 2ddition of 30 kGy and 100 kGy tre~tm-nt-. The F~-IR r--ult~ are gi~en in ~able 1.
3S Table 1 - The E~fect of Varv~ the Irradi~51s~ L~_L3~ Azla~Q~e - Concentration on the ~atio of ~he Azl~ctcne-to-PE IR S~gc~l~

F~-IR Ratio (1824s1462 cm-l) at Variou~ IrradLa~Qa Dgae~ ~kGy~
DOB - ( k~y~) - ~ 30 50 1QQ

- 0.38 1.70 1.32 5.06 4S 100 1.10 5.23 0.96 W093/25594 - PC~/US93/04~55 ~
213~00~ -The marked ~) me~brane wa~ completely filled with polymer and swelled upon solvent rin~e Upon dr-yinq it wa~ too thick for accurate IR measurement; thus the ratio i3 not indicative -of the amount of grafted VDM
A ~urface area measurement was performed ~ccording to tho following method A sample measuring approximatsly 3 cm X S cm wa~ pl~ced in a tared ~ample holder of a Quantasor~ BET Surface Area Analyzer (Quant~chrome Corp ) The sample wa~ dega-~ed by flu~hing with helium at 50C for 1 hr The sample holder wa~ then immer~ed in liquid nitro~en, and a helium~Xrypton gas mi%ture wa~ pas-ed through the sample At thi~ te~perature, only the Xrypton w$11 adsorb onto the surfaces of the ~ample, thus depleting the X~ypton in the ga~ mixture pa--ed through the ample The ~urface are~
calculation i0 based upon the A~sumption tbat th0 prob~ ga~ ad~orbs lS on all avail~ble sample su~f~ce area in a monolayer thus ~he amount adsorbed times the ad~orbate cros~-sectional are~ i~ proportional to ~he pecific surface area The depletion of Xrypton from the mixture, (i e , the u~ount of Krypton ad-orbed) is det~cted with a sen-itiv~ thermal conductivi~y det0ctor; Upon rewanming of the sa~ple to room temperature, the adsorbed Rrypton is s~l~a~ed and also guantif~ed She amount of ad-orb~d Xryton and th~ mas- of the sample are u~d to calculate th~ pectific surface area/unit mass value Th- abovo 8ET urfac~ aroa m~a-usement wa- perfonmed on 2S the unirradiated hy~rophilic membran~ control and on th~ 50 kGy-100 ~a~pl- showed that tho control value (18 6 n~lg) had b~n reduced by ov~r 50~ to 8 1 m2/g by the grafting of poly~inylazl~ctone), which i- not deQmed to be diminishod ~yond usefuln~sJ o~ th- m~mbrane In all ~nst~ncQ- but on~ ~30 kGy/50%), thera wa- no -d~-ctabl~ welght loss followLng the xt-nd~d 301~ent extraction - - -descri~ed in Exampl~ 1 Of thc po-~LbLl~ti~s, the proces- employing 50 kGy/25~ med to be tho best co~promise to avoid pore blockage while providLng azlactone functionality ~ - +

3S ExamDle 4 - R~action of ~lacto~Çrafted HvdrQPhlLi~ P~ ~embrane wLth AmmonLa Port~ons of the treat-d m ~brane~ d~scrLb-d ~n Examplas 1 and 3, each prepared uslng 50 ~Gy and 50~ VDM, wor~ placed ln an ummonia atmo-phere in an enclo~ed gla-- ve---l by u-p ndLng them- - - l a~ov~ a concentrat~d N~,OH olutlon for 10 mLn at ambi-nt ~emp~ratur- Fr-IR (usin~ a Mod~l FTS-40 pectrophoeometer, ~io-Rad, Digil~b Dlv , Cambridg~ ) mea-urOEm nt~ of both a~00nla-r~act-d ~ mbran~ and unr-act-d control m~mhran~ how~d that the 1824 cm-1 azlacton- ab~orbance b~nd on the u~mo~ia-react~d membrane~~
4S had n~arly ntLrely ~L~appQared and that a new band appQar-d at 1659 ,., 1 ~-' '~ '' '' WO 93~25~94 PCI'/US93/04~5 cm-l which i~ indlcative of an amide bond Thi~ ~onflrmed that virtually all of the azlactone i~ available for reaction The~e r-~ult~ ~how that almo~t any type of ourface che~i~try might be prepared from an azlactone-grafted membrane ~urface by choo~ing a~ a seeondary reagent one which ha~ both the de~ired functionality and an amine functionality Exam~le S - E-beam-Graftin~ of Azl~ctone to Porous Polvethvlene ~i~m~ Increase~ the_~oLnt of Cou~led Protein Azlactone-functional and ungrafted con~rol m~mbrane~
w re propared as de~cribed in Example 3 Protein ~olution- wer~
radiolabeled using Iodo-Bead~T~ bead~ (eommercially available from Pierce Chem , Roc~ford, IL) and NaI-125 (Dupont NEN, Bill-rica, MA) u-ing th- procedure- do~crib-d in the product in-ert Sp-cific radioactivitie~ obtained were Protein A, (Genzyme~ Bo-eon) 2782 cpm/~g; immunoglobulin G ~IgG, Sigma Chem , St Louis), 2000 cpm/~g;
and bovino ~-rum albumin (BSA, Sigma), 2885 cpm/~g Circular por~ion- (~ mm diameter) of the membrane were eut out using a paper punch The membran- diw ~ were then incubated with radiolab-led protein in 250 ~l of 25 mM ~odium pho-phat~, 150 m~ NaCl, pH 1.5, for 60 min at ambient temperature So~e m mbrane~
wer- r-acted with 3 0 ~ ~thanolamin~, pH 9 0, for 30 min prior to th- protein incubation to ~deactiv~te~ th- azlaetone functionality Following th~ protein incubation- all meobrane~ were r-acted an 2S additional 15 min with 500 ~l of th- th~nolamine r-ag-nt eO
inactivate r~maining azlaetone~ a- well as rin-~ out unbound prot-in Each mcmbran~ wao-~ub--quently rin-~d an additional three t~mo- with 500 ~l of the phocphate buffer After the bound ~ radioactivity wa~ det-rmined ucinq a ~od l 5230 Auto-Gumma ~ 30 ~cintillation counter (packard~ Downer- Grove, ~), th mQmbranes w~rn incu~atod for 4 h at 37 C in 500 ~l of l 0~ od~u~
dodecyl~ulfat- ~SDS) ~olution followed by determination of r~ridual ~-~ radioactivity SDS i- a ~trongly d-n~turing det-rgent capable of --- -- romoving all but th mo~t t-naciou-ly bound protein ~n the-~
expor~m-nt-, control m~mbrana- wor- compl-tely untr~at-d Th--- and all experiment- dQ~cribed ~n thi- ~xampl- were p~rfor~Qd in triplicate .

.

W O 93/25~94 PC~r/USg3/045~
23~0'~ 1 ~able 2 - The Bindlnq of Three Protein~ to E-beam-Gr~ftçd a~d Control Porou~ ~embranes --Total 8Ound Coupled Prot~in SDS Protein Membrane ~c/çm~) Resi~t~aCe~%L (~atçm~) Wlth Protein A
Untreated 4 0 14 0 55 Untreated Quenched 4 2 11 0 50 Grafted 3 2 S8 1 86 1~ Grafted Quenched 2 7 21 0 57 With ~m~uno~lobu~in G
Untreated 7 2 18 1 24 Untreated Quenched 8 9 11 0 95 Grafted 6 1 4S 2 74 Grafted Qu~nched 4 8 27 1 31 Wi~h Bovine Serum Albumin Untreated 3 4 18 0 59 Vntreat-d Quenched 3 6 15 0 53 Gra~ted 1 7 68 1 18 Grafted Quenched 1 4 31 0 42 !
Con-i~tently for all three pro~ein~ there wa~ at least a two-~old incroa~e in the amount of couplod protcin a~ a re~ult o f th~ e-beam/VDM treatm nt Thi- wa~ e~pecially ~urpri~ing bQcau-e th re i~ a decr-a-Q ~ total ~inding The cau~o of the d~erea~e in total binding wa~ unc-rtain ~ince thi~ aerie~ included no e-beam only or -b am plu~ ~olvont control-; how-ver, it wa~ pr~umed that the 50~ r-duction in total urface arQa ~compare with ~xample 3) i~
the r~a-on --Tre~tment of the membrane ~ith ethanolamine to inactivate the azlactone funetionality reduced the amount of coupled protein to about the level of untreated membran~
.
xam~le 6 - The Bindinc Qf Radiolabel~d Pro~el~_to ~-8~am Grafted Porou~ Polyethvlene~ brane ~5 ~ Unlos- indlcated otherwi~-, all graft~ng proeQdure~ ar~
idanticaL to the 50 kGy xample d-~cribod in Example 3 ~mbrane~
wer- treated a- d--cribed in Exampl- 5 Protein A pec~c radloactivity wa- 1884 cpm/~ mbran~ w-r- incubated ov rnight (16 h) w~th continuou- rocking and treatm-nt with th nola~in~ wa~ _~
SO incre-~d to 50 min Control~ wer~ -beam-trQated only and -b-am-tr~ated plu~ ~olv~nt-tr-ated W O ~3/25~94 2 1 3 6 0 ~ 4 PC~r/U~93/045~5 -: Table 3 - B~nd~na Protein A to E-beam Grafted a~ld Cont~ol_PorQu~ Membrane~
Total Bound Coupled S Protein SDS Protein Membrane tu~/cm2) Resistancel~) E-b~a~ 9.1 33 3.0 E-boam I Solvent 7.9 25 2.0 E-beam ~ 50~ YD~ 7.; 56 - 3.9 E-beam ~ 100~ VDM ?.~ 85 6.4 Th~ ~olvent treatmont caused a 13% decrea3e in the Amount of protein which bound to an e-beam-treated membrane, because of the Aolvent ~olubilizing ~ome of the hydrophilizing hell of poly(vinyl alcohol) which i9 requised for tha membran~ to wet.
- Ther~ wa~ no significant effect of azlactono on the ~QS~l binding twhich i~ probably propor~onal to th~ to~al av?ilable surface area); however, th~r- wa~ a pronounc~d effect of azlactone on th~
qualLty of the protein binding, i.e., a large inor ase in th~ ~mount of th~ prot~in ~hich i9 ~0 tightly bound,i-e-, co~plad, that it re-int~ removal by SDS. Although it i~ not pr~ci~ely accurate to a~cribe covalency to tha SDS r~istant fraction, it i~ hiqhly probable that increi~e~ in 5DS re-i~tance refl~ct increa3es in 2S co~alent binding.
Al~o, increa~inq the incubat~on time for the protein binding step from 1 h (Example 5) to i6 h lncr-~ed the umount of the total binding 2.5-fold and th~ amount of coupling 3.5-fold.
Thu~, a 1 h incubation did not allow for full, but pas~iYe diffusion of the protein through th~ membrane.

~ ~ ~ Exam~le 7 ~_Effe~t of VD~LÇon~ent~tlon o~ Psotein aind~q Ne0brane~ wer~ $rrad;at~d wi*h 50 kGy and treated as de~cr~bed in Exa~ple 3 and protein coating proc~dure~ w~re identical 35- ~to tho~ in Example 6. Protein A pecific radioactivity was 1767 cp~/l~q ~

TablçLiL ~t ~ect of VD~_Çoncentration_~n Protein 3~ndin~
Tot~l ~ ~ gO- Bound Coupled - Prot-in SDS Prot-in S~
- MRmbrane~u~lcnr)Re l~t.(~) ~ ) Ratio~
--- E-~ ~m 7.53 25~ 1.84 0.0 - _ ~ 45 ~-b am + ~ol~-nt5.97 21 1.47 0.0 2-b am 1- 25~ VDM6.55 30 1.97 0.168 E -b~a~ + 50~ VDM 6.19 36 2.20 0.638 E-~eam +100~ VDM2.58 67 1.73 0.540 SO ~Ratio of the ab~orbanc- of 1824 cm-1 azlactone band to 1462 cm-l polyethyl~n~ ba~d.

W O 93/25594 P ~ /US93/045.~5 21~'.i(3~

There were two effect~ of increa~ing VDM concentratio~
1 ) A concentration-dependent increa~e in the percent of CDS
re~i~tant protein; 2) a eoncentration-d~pendant d~crea~e in the total prot~in binding These effect~ combine to yi~ld a~ optimum S amount of coupled Protein A at S0~ VDM Sh~e oppo~ing effect~ were con~i~tent with raduced accea~ of protein cau~ed ~y exceedingly long chain~ of poly(VDM) at high YDM concentratlon~, blocking protein acce~- ~o the inner membrane ~urfac~

Examole 8 - Retentioa o~ Bio~o~ical Activi~v of Mg~bran~ und rotein It wa~ found that Protein A bound ~o~al8ntly to the porou~ PE msmbrane through azlactone reta~ned it~ abllity to bind ~5 human IgG Thi~ wa~ accomplished by a two-part experiment determination of Protein A b$nding using radiolabelæd Protein A, and, in parallel, de~ermination of the amount of radiolabel~d IqG
bound to membrane-bound unlabel-d Pxotein A
~embrane~ were prepared a~ de~cribed in Ex~ple 3 All binding procedure~ w-r- identical to Example S except that incubat$ons with Prote$n A ~whether radLolabelad or not) wer6 for 5 5 h~ Radioactivity determination~ were ~ade on tho~e ~embrane~
which had been incubated with radiolabeled Protsin A (1590 cp~
~pecific radioactivity~ Sho-- m~mbrane- which had been bound with unlabeled Protein A wer- incubated an additional 16 h with radiolabeled IgG (~pec~fic radLoactivity 1500 cpmt~g) They were rin-~d, and IgG ~inding ~# dot~rminod by i~otopic docay followed by the SDS stQp and a r-p ~t of th- binding det-r~ination ThQ re-ult~
from thic eri~ of x~oriment- ar~ found in T~ble 5 ~h~ oontrol 0embrane~ wer0 e-~ am- and colvent-tre~t-d Table 5 - The Bindinq of $cG to ~otein A-~ou~led Membrane - - -__P~ote~n A _ lTmun~qlobuL~ Ç

3~ ~otal Total Protein A SDS IgG SDS
Bound Re-i~t Bound Reci~t Membrane~uo/~m2) (~ ~u~Jcn~
~0 Control 7 66 20 12 1 2 9 ~ Control 1 25t VDM 6 84 31 10 6 3 4 Control + 50t YDM 5 63 42 9 9 3 5 Control +100% VD~ 3 03 57 3 8 4 4 Sh~ higher SDS rei~1-tanc-c for the coupling o Protein A
~o the grafted membran- (compar-d to the b~nding of IgG to the W O 93/25594 2 1 3 ~ ~ il PC~r/US93/045~5 Protein A-deri~ati~ed, orafted membrane) showed that Protein A was bound covalently to the membrane and IgG was bound non-covalently to the Protein A The SDS resistance~ of 3-4~ indicate that the VDM
wa- almost complet~ly inactivat~d A~ ob~erved in previou~
S example~, optimal ~ovalent binding wa~ at 50~ VDM

Examole 9 - The Time-Cour~ for the ~indin~ of Protei~ A to VDM-Grafted Membrane~
Tabl~ 6 ~ummarize~ the re-ults of ~e~ral previou~
exp-riment- in b'nd$ng Protein A to grafted PE membrane~ in which length of incubation of the protein with th~ mombranq wa~ varied Although the experimental condition~ were not xactly comparable, ehoy ~howed a tr-nd that prot-~n binding i~ highe~t if allow~d to procoed ovornight (16 h~ An approximation of overnight b~nding can be obtain-d within on- wor~day by incubatino for at least 5 5 h Table 6 - Time-Deoendence of the Rindina of Protein A to VDM-Grafted Membrane Protein A
~ Ex~mDle Time (h)3Ound luo/cn~
; 4 1 3 2 ~- 2S 7 5 5 ` 5 6 6 16 ~ 6 2 Sh--o r--ult~, obtained from pa--ive diffu~ion of prot-in into th m~mbrane, d mon-trat~d a definfte time-dep ndence ~ A dr mat~c wcal-ration (perhap- a hundred-fold) would occur if protein wer- drawn ~nto the membrane through ~ome activ- proce-~
- uch a-~lioht pre--ur ~diff-r-ntial Exa~o 10 - The Eff-ct of Hioh Salt Concentration on Btndinc Protein A
~0 ' U~ing the teaching~, p clfically Example~ 1-35 of U S
Pat No 5,200,471 (al-o publiahed a- PC~ Publicatlon WO 92/078~9), ~- - lt wa-~ inv ~tlgatod wh-ther high ulfat- concontrat~on could al-o nh~nce th- binding of protein to azlactone whi~h had been E-beam ~ - ~ graft-d to PE membran--4S Prot-in A w-- incubated w~th ~ar~ou~ me~br-ne- or 19 h in 25 mM odium pho~phat~, pH 7 5, 150 mM NaCl (low alt) In the WO 93~25594 PCI'/US93/045~5 2 1~3 ~ 0 C ii "high salt~ incubation 1.5 M sodium sulfato was substLtuted for the ~odium chloride. Control~ were e-be~m- and ~ol~ent treated.

~able ? - The Effect of Sodium Sulfate on Protein Coue~lng to S Grafted Mem~ranes Ratio of ~igh Salt~ to ~ow sa lt~ Bindin Total ProteLn Coupled Bound SDS Protein ~a~hL3~ (uq/cm2)Re~i~t.t%~ tuqtcm2) Control 9.5/5.3 30/20 2.8/1.0 Control + 25~ VDM 10.5/S.8 45/30 4.811.8 Control + 50~ VDM 9.4/4.9 55/34 5.1/1.6 Control ~100~ VDM 6.6~2.7 70/58 4.6/1.5 The re~ulta are quite con~ ent with thoae ob~rved with Protein A on hydrophilic azlactone-functionalized porou~
polym~ric bead- as zhown in ~xamplo~ 1-35 of Application 07/609,436.
Ther- wa~ 2.5-3-fold increa~e in the amount of coupled protein, a 75-150~ increas- in total ~inding, and an increa~e in S~S r~ tance 3 of 20-50~. Additionally the~e experiment~ confirmed conclusionn drawn in earlier exp~r~ent~: optimal VDM concentratio~ i8 1-B~
th~n 100~ and perhap~ about 50~; the pè~centage of the bindlng which i8 SDS re~i~tant continued to incro~e in proportion to the p~rce~tage of azlactone.

Exumsle ll - Azlactonç-Gra~ed Memkr~ne~ are ~ ul 1~ an - - ;
Immunodiaq~o~tic One of the major u~c~ of ~pecialized, biocompatLblc m~mbranos in biotechnology i~ to ~mobllize one of the reactant~ in a elinical diagno~tic te~t a- in a het~rog~neous E~ISA-type a~ay.
See for example, European Faten~ Publication 0 294 105 (Rothman et F
al.). In this example, it wa~ demon~trated that an azlactone-functionalized mambrane could b- u~ad to ~ind an ant~body and that 'the resulting der$vatized membrane could be u~ed in a chromogenic E~tSA ~en~yma linXed immuno~or~-nt ~-ay). ~ -Strip- of azlactone-functionalized m-mbrane~ prepared ;~
- acco~ding to Ex~mpl~ 3 above wer- $ncub~ted with con~inuou~ rocking for ~7 h at a~bi-nt t~pesatur~ with ~ith~r human IgG ~hIgG in 100 ~~
~M NaCl and 100 ~M odium pho-phata olutlon, p~ 7.25) or bovine ~eru~ albumin (BSA),.aach at 1.0 mg/ml, in 10 ml of 25 m~ sodium phosphate, 150 mM NaCl, pH 7.5. They were then given sev~ral 1 h rin~ in buffer, dried, and stored, desiccated, at ambient temperature until u~ed. Prior to use, to in~ure ~hat all azlactone-_ W O 93/2S~94 2 1 3 ~, O O q PC~r/US93/04555 funetional moieties were reacted, mem~rane disc- were incubated with 3 0 M ethanolamine and 1 mg/ml BS~, pH 9 0, for 30 min , rin-ed and u~ed in the a~ay de~cribed below The a~ay was initiated by incubation of the discs with S 10 ug/ml anti-human IgG-HRP ~hor~-radiah peroxida~e~ conjugat~
~Cappel-Worthington, ~alvern, PA) for 1 h with continuous rocking Thoy were rin~ed for 4 h, with rocking, with PBS-Tw en (25 mM ~odium pho-phate, 0 6~ Tween 20, pH ? 5) and transferred to clean te~t tube~ for a chromogenic HRP sub-trat~, o-phenylenedi~mine ~Sigma Chom Co ~ ~3 mM in 100 mM sodium citrate buffer, 0 12 ~g 30%
hydrog~n peroxide, pH 5 O) The product of the perox~dation form~
an orange-colorod, partially-in~olubl- product after reaction with 2 5 M H~S0, Sp ctrophotometric e~timation- of tho reaction were obtainod by transferring 50 ~1 of th- reaction ~up~rnate to a lS 96-well microtiter plate containing 20 ~1 of 2 5 M H~0, Re~ult~ of a~sorbance det~rmination~ at 4gO nm on a microtiter plate ~p~ctrophotomoter (Dynatech, Chantilly, VA) ars given in Table 8 .
Table 8 - Com~ari~on of the Bindin~ of Anti-IqG-HRP to Control and IsG-Contalnlno Membran ~

8RP Acti~itv /mAe49Q/min) IgG/BSA
- ~ Samol~BSA-TreatedToG-Treated Ratio 25~ VDM~ 98 451 4 9 50~ VDK60 201 3~4 lOO~ VD~41 199 5 5 - - - In ach ca-- t~er~ i- con-id~rably mor~ acti~lty a~-ociated~with the antibody-cont~ining membranes than with the BSA
controln ~ I
3S- ~ ln thi~ exampl~ a 150,000 dalton ant$body wa~ ¦
L~mobil$2~d, th-n complexod with a 200,000 dalton antibody--n~yme conjugate, ind$cating that th~re i~ not a gr-at barri-r to working with l-rg- prot-in comploxeJ
-~ Exam~le 12 - Gamma Irrad~ation Çraft~no of ~Y~ç~hili~ed MicroDorous - ~ n~m~ran- w~th VDM ~n~ HEMA
~-~ 15 pr-w igh d pi-c-- of PE oicroporou- ~ombran-, having _- a thln rh-ll of poly(~inyl alcohol) prepar d according to ~x mple 22 -- _ ~ of co~--ignod, copend~ng application 07l775,969 (Gagnon et ~ ndPCT Publlc-tion WO 92l07899, xc-pt that PE wa- u-~d in-toad of iP, m a~uring 7 6 X 20 3 cm, w r- rolled-up and plac~d into gla-s ampul-- ~h- ampu~e- wor- vacuat-d to pr--~urs- l-s~ than 2 x 10 mm Hg and th~ gla-~ n~cX~ w-re melt--ealod to pr-v-nt 2 WO93/2~594 . - PCI~US93/04 i5 213~0~

contamination $hree additlonal piece~ were placed in unsealed test tube~ All 18 ~ample tube~ were expo~ed to gæmma irradiation for -=
9 5 hour~ for a total do~- of 38 kGy~ ~he tubes had b~en placed ~ide-by-~ide i~ a large envelope which wa~ configured normal to the ~ource, ~o that they would all b~ exposed to the ~ame do~e ~he envelope was rotated 180 after about the fir~t 4 hours of irradiation to further en~ure that the~sample~ were do~d evenly After gamma irradiation, the tube~ were pla~ed into a glove bag, which al~o contained the argon-purged (i e , 0~-free) monomer ~olutions li~ted in Table 9 below The glo~e bag was flu-hed with argon by 5 inflate/deflate cycle~ to remove a~ much 0 as po~ible Four ~ealed ampuie~ were broken open, and the film samples within them were placed in either pure ethyl acetate (EtOAc), 10 wt/vol ~ VDM in EtOAc, or 10~ VD~/25~ ~EMA in EtOAe, ~5 r--p ctively, Th -e wer- allowsd to ~oak for longer than 5 minute~, before being removed from the olution and ~tored in ~toppered te~t tubes A total of thre- amplo- were pr-pared for each monomer ~olution ~fter all reactions were complete, the ~ample- were removed from the glove bag and rin-ed 3 time~ in fre~h EtOAc to remove exce~c monomer, and air dried ~ he m~brane- wor~ analyzed for grafting add-on by weight uptak and by FT-IR cpQctro~copy The weight uptake data sh~wed that the 10~ VDM and 10~ VDM/25~ HEMA me~brane~ av-raged about 0 7 and 1 0~ wt, uptake Th- IR spectra confirmed the wt uptak~ data, ~howing ~ignificant ab-orbanc- at 1824 cml for W ~ in both th 10~ VDM and I0/25 VDM/HEMA ~ample~ Tbe ~ample- al-o display-d an ab-orbanc- at 1670 cm~, indicative of partla}
hydroly~L-~of the VDM moiety An additional ab-orption peak at 1728 cm', for tbe 10~25 VDM/HEMA ~a~ple~ confirmed the incorporation of ~ th- HEMA monomer into th~ grafted copoly~er No 1728 c~ peak could b- een in the 25% HEMA m-mbranes The wt upt~ke data4an~ the ab~orbance value~ for tbe VDM and ~EMA functionaliti-s, nonmalized to th~ PE ab-orbance p~ak t 1462 cm~, are t~bulated belo~
Tabl- 9 3S ~ ~5~:~Y~
AVE WT~ 1824cm'/ 1728cm~/
~M~E ADD-0N 1471cm~ 1471~n~' Control 0 000 0 000 0 000 ~0 only 0 115 0 000 0 000 - -~ ln air 0 160 0 000 0 000 _-~ + EtOAc 0 160 0 000 0 000 - ---~
y + 10~VDM 0 718 0 060 0 000 ~ + 10VDM~25HEiMA 1 013 0 040 0 120 SO

~ . ~

W 0 93/~SS94 2 1 3 ~ ~ ~ 1 PC~r/US93/0455 SummLng the IR ab~or~ance ratioo (inclu~ing the 1670 cm' peak) gives an indication of overall add-on.

S ExamPle 13 - ~-be~m Irradi~tion Graftina of ~vdro~hiL~ed Micro~orou~ Mem~ran~ with VD~ and ~E~
Pre-irradiation e-beam grafting of hyd~ophillz~d PE
microporou~ m~brane~, prepared ~ccording to Exa~ple 3 ~bove used the same equipment of Exampl~ 1 akove, except that modification~
were made to th~ glo~e box to mini~ize prn~encs of ~. An ~
analyzer, in~talled in the glove box to monitor the ~ concent~atisn during the run, ~howed that the~e L~provement~ allowed th concentration to be maintained at les~ than 30 ppm - often a~ low a~
10 pp~.
Piece~ of the membrane wer2 tap~d to a polyethyl~ne terephthalate (PET) carri~r we~ and pa~ed through an e-beam curt in at 6~1 m/min. Th- e-beam accelerating voltag~ wa~ ~et at 150 XeV, and a dosa rat~ of 50 ~Gy- was used to irradiat6 t~ m~mbrane~. ~he ample~ came out of the e-b-Am cha~ber diroctly into ~ N2 purged glove box where they were immer-ed ln the monomer solution. The inert at~o~phere help~d to pr~vent quenching of the genera~ed radicals with oxygen.
~he ~olution~ had concentration~ (in wt ~) ~DH and HE~A
in ethyl acetat- in concentrationr 1~ ~ted ~ n Table 10 below.
2S Irradiated ~embran- ~a~ple- were ~o~kod in the monomer solution for 24 hour-, follow~d by a thro- 5 m~nut~ soak~ in fresh othyl ac-tato in ordor to wa~h out xc-s- ~ono~e~. Th~y wer~ then dried and placed ~n zip-lock typ- bag- to pr~vent pos-ible hydroly~i~ of the aslacton~ by at~o-pheric w~ter.
~he membraner were incubatod Sor 16 h with ProtRin A at 2s0 ~gfml in either 25 m~ or 500 mM odium pho~phat2 buffer ~pH
7.50) which wa~ ruppl~m~nted with 150 mM N~CL or 1.5 M ~odium - ~ ~ulfate. ~h~ ræsultD are pre~nted in ~abl~ 10. ~hos~ ~xp~rLment~
perfor~ed in the 25 M~ buff~r ar- indicat~d by an a~teri~k. All r~rults ar- the average~ of tripl~cat~s.

, . .
_ . :
,

4 PCI/US93/04~C~5 2 13 ~ O ~ L

, able 10- Effe~ts of Graftlna HEMA in Combination with VDM into PE
,Membranes Initial SDS Coup~-d Sample Bindin~ Re-i-tanc~ B~nding S ~VDM/~EM~) (u~/cm ) (~ (u~/cmf) Salt Cl SO Cl ~5~ Cl ~5L
Untr~ated 10 66/ 9 78 8/10 0 81/ 0 97 Solv-nt ll.S7/10.29 21/31 2.38/ 3.18 E-b am~ 11.97/ 9.52 23/30 2.72/ 2.86 E-b am 10.59/10.13 23/31 2.44/ 3.09 0/ 2.5~ 0.43/2.02 27 /36 0 11/0 73 0/ 6.5~ 0.3Ç/1.41 28/34 0.10/0.49 lS 0/10 0.27/1.81 24/42 0.06/0.74 0/12.5 0.80/3.95 25/38 0.21/1.45 0/25~ 0.30l1.07 28/32 0.09/0.36 0/25 0.26/1.01 24/38 0. 06/0 38 ', 10/0~ 4.06/5.14 29136 1.19/1.85 10/0 4.3317.21 29/42 1.25/3.05 - 25/0 2.6813.58 36/42 O.9S/1.47 25/0 2.73/4.30 38/4g l.OS/2.11 S0/0 2.78/3.65 77/84 2.14/3.06 lQ/10 3.57/ 8.96 SÇ/81 2.01/ 7.28 10/25~ 2.10/ 9.70 67/94 1.52/ 9.10 10/25 0.82/15.55 S0/9S 0.49/14.72 ' ~ 25/2.5~ 2.45/4.53 54/74 1.33/3.35 - , 25/~.25~ 2.69/5.~8~ 82/89 2.14/5.11

5/12~.5 S.10/8.98 Sl/78~ 2.62/7.01 25l25~ ~.48/9.82 63/92 0.93/9.04 50~10~ ~~ 1.8S/3.3069/77 1.2~/2.56 Microoorou--Monbrane ,, , A pi c- of hydrophilLc PE m mbrane prepar-d in th~ ame 40 - aDD r~,a~ for ~xampl- 12~ aDov- wa- oak-d_with an ethyl ~c-tat-olution of-~25~wt~/vol ~ VDH~ in -thyl ac-tat~ ~ 0.25~ uv initiator azobi~ obutyronieril-) ~comm rcially available a- Irgacure~ 907 from~c$ba~G-igy) ànd th-n fed into a N~ purg d RPL uq troater at 7.5 m/min.~--t~at~21 -mp lamp pow r ~310kW/ms). Anothcr ampl- wa----,- 4S ~ traaé-d th ~ m ~way, ~-xc-pt tbat th monom-r olution al-o conta n d 2.5 wt ~ cro--lin~er~n op ntylglycol diacrylato, NPGDA).
Som vaporation o~ th monom r ~o~ution did occur prior to, and ~' 'during the irradiation.
~R p ctro-copy how~ that VDM did inde-d b co~e graft-d onto th- membran- -urf-c-- in both ca--~. U-ing eh rat~o ' i~
of th- R b-orbanc- for VDM at 1824 cm~ to th ab-orb-nc- of PE ~t 1462 cm'l a~a ~a-ur- of VDM add-on, howed that,the amplo without th~ NPCDA had gr--t-r add-on than that with the cro--liAker;

' ::

W 0 93~25~94 2 1 3 ~ P ~ /US93/045~5 ExamDle -~5- Pre-Irradiation EB Graftinq onto H-PP Membrane__and PE

Hydrcphilic polypropylene ~pp) membrane wa~ prepared in the m~nn~r according to ~xample 22.of coassigned, copending U.S.
Patent Application Ser. No. 07/775,969 (Gagnon ~t al.) publiHhod a~
PCS Publication WO 92/07899. PE blown microfiber (BMF) web wa~
prepared according to Example 19 of Gagnon et Al.),to become a calendared BMF nonwo~en made ~rom Dow 6808 LLDPE reain ~t a ba~i~
wt. of 94 g/m.
All ~ample~ of PP membrane and PE BMF nonwoven were irra~iated in the manner a~cording to Example 13 above with 50 ~Gy~
o' 150 XeY e-beam irradiation prior to i~n~r~ion in th~ mcnomer ~olutions li~ted in Table 11 in a <30 ppm 0. atmo~phere. All monomer solutions, in ethyl aoetate, had ~een purged with argo~ to 15 remove 0R~action wa~ allow~d to proceed for about 5 minut~
prior to removal and rinsin~ in pure thyl acetate.
The tablo below lists the gr.afting wt. ~ add-on, expre~ed as-final wt - initial wt 2~ ~ 100 in~tial wt for the sample~ in term~ of the monomer solution u~e~.

Table 11- E-beam Gr~fted VDH on ~P ~q~ane and PE BMF
W~51GF~ PEJlOE~T . A~ -ON
PP-Membrane ~-8~F
10~ VDM 23~ 15%
25~ HEMA - 265~ 328 ~ ~ 10/25 VDN¦HEMA 215~ 136 ~nfr~red ~pectroffcopy confirmed that the-e monomer~ w~ra indeed incorpora~0d onto the surSac~ of the~e ~ub~trate~ ac grafted polym rs..

ExamDle 16_- Pre~ar~ion of ~n~ Pro~ein 8indin~ Q~ ~la~ma VDH-~rea~ed Materi41~
~ydro.2hil~zed, porou- polyethylono (PE) mambran- i pr~pared according to Example 12 bove wa~ u~ed w~thout further tr-atmont.- Non-porou- f~Lm~ of polypropyl-n~ ~bi~xially ori~nted, th-r~ally ~xtrud-d, 0.05 ~m th~ck PP film)~
poly~eth~l~n~tsrephthalate) (b~axi~lly orientod, 0.1 mm thlck, PET
film) and poly~tetrafluoroethyl~ne) (0.05 m~ thick PTFE film) w~re u~d witAout additional tr~atmont.
Vinyldimethyl azlactone ~VDM; wa~ depo~it~d onto ali the ~5 fil~ ~ample~ ~multaneou-ly ~n a glow di~charge.- Th~ glow discharge depo~ition~ oc~urred in a belljar v~cuum ~ygtem u~ing two parallel W O 93/25~94 - PC~r/US93/04S55 213~ G 'd, plate ~lectrodes ~20 cm X 30 cm) ~paced 5 cm apart The film amples were placed on the lower electrode ~grounded) She material~ were ~ubjected to a VDM glow di~charge at 60 mtorr VDM
pre-sure with a 15 w di-charge power generat~d at a frequency of 25 S kHz Fir~t one side waa treated, then the ~ample~ wer~ turned over on the bottom electrode to treat the other ~ide The nominal thickne~- of the depo~ition wa~ 70 nm on each ~idQ of ~he ~ample~, a- mea~ured by a quartz-crystal-microbalanc~ ~xposed to the di~chargo during the depo-$tion Alt-rnatively, the film ~ample- were given an in$t~al nitrogen-containins aurface by nitrog-n dLccharge prior to VDM dopo~ition (noted in Sable 12 below as ~N/VDM~ tr~atment~
Prior to glow di-charge depo~ition the ample~ were treated with nitrogen gas ~200 mtorr) glow dLscharge of 15 W ~25 kHz) for lO g Thi- wa~ followed by the VDM treatment a- de~cribed above Triplicata ~8 ~m) di~cs of each material wero cut using a tandard office pap~r punch and p}aced in 2 m~ micro cen~rifuge tubo~ followed by addition of 200 ~L of the buffer ~olution containing radioiodinated Protein A ~ranging from 2200 to 2500 cpm/ug of protein) Th- chloride buffer con-i~ted of 150 mM NaCl nd S00 mM od$um pho~phate, pH 7 5; th- ~ulfate ~uffer wa~ l S M
odium ul~ate and 500 mM sodium pho~phate, pH 7 5 The disc~ were incubat d with tbo olutions for 17 h (with continuou~ rocking) to allow the protein to fully eguilibrato throughout the porou-2S ~embrane The coupling reaction wa- topped by addition of S00 uL
of 1 0 M ethanolamine ~in 25 mM sodium pyrophosphate, pH 9 o)~ twice for a total of S h After thr~ additiona} wa-ho- with the chloride - buffer the di-c- were tran-f-rred to anoth-r to~t tubo, and the ~--ociated radioact$vlty wa- detorminod ucin~ a Packard ~odel 523Q
- - 30 gamma rad~ation detector -~ Protoin which wa~ re~i-tant to olubili2ation by a troat~ent with the protein denaturant sod$um dodecyl~ ~ulfatQ (S2S) was op~rationally def$ned a~ ~eovalently eoupled~ to the ubstrate - - -- Thi~ troatmen~ wa- with a ~ SDS colution (in 25 mM odium pho-phate 3S buffer, pH 7 5) fo~ 4 h at 37-C, followed by thr~e washing- with the warm SDS ~olution, and ro-determ$nation of th- u~ount of as~ociated ~' radioactiv$ty ,, , . _ -- -. ~

W O 93/25594 2 1 3 6 0 0 i P ~ /US93/045S5 Table 12 ~ æCo~plina ~_P~otein to Pla~a-~r~ated Material~

Proteln SDS Protein Bindin~ Re~i~tanceCoupl~ng S Materi~L Treatment t~3/~m~ 515~) Cl ~50. Cl /S~a PE Control11.2/ 9.5 33~343.7/ 3.2 VDM 7.6/12.0 6a/755.2/ 9.0 N/V3M7.9/20.6 79/83~.2/17.0 PP Control0.6/ 0.4 21/170.1/ 0.1 VDM 0.2/ 0.5 42/540.1/ 0.3 N/VD~O.g/ 0.7 75/690.7/ 0.5 PET Control1.41 0.8 27/200~4/ 0.2 VDM 0.5/ 1.4 55/700.3/ 1.0 N/VDM0.4/ 1.1 62/650.2/ 0.7 PTE~ Control0.4/ 0.3 22/1~0.1/ 0.1 VD~ 0.2/ 0.7 38/720.1l O.S
N/VDH0.3/ 1.2 56/760.2/ 0.9 Comparing just the Co~rol ~amplea, much more Protein A
bind~ to the PE me~brane than to nny of the fil~s. Thi~
undQr~tandable becau~e the membrane ha~ about ten-fold morR total ~urf~ce area th~n th~ f~lm~. Shu~, it is quite surprising to ob~orve a two-fold increaae in the init~al binding resulting from tho nitrogen/VDM t~eat~ent, ~ince the nitro~en treatmont and YD~
depQsltion do not pen~trata ~ub~tantially into the pore~ of th~
mQmbrane. S08 Exa~ple 19 below. Actual enhancQ~ent of prot~Ln binding to PE i8 clo~er to th~ 400~ ob~r~ed fo~ the PTFE.f~lm~
than the 30~ ~oen with the PET film~. Such a high enhanc~m~nt f~ctor on PE ~embrane- maans that one mlght ~ak~ a ~Lngle-layer ~embran~ act like a laminated, multi-layor~d Dembran~ by s~Or~ace treatment on on~ id~ to pro~uc~ lay~r A, followed by trsatment on the other ~ide to produc~ lay~r C, follow~d by treat~ent w~th an e-beam or other penetr~tlng actlvator to produce layer B. Lay~ra A, B, and C might repro~nt thr-- different grafted ~ono~ers, conferrin~, ~.gO, different hydrophllicitie~ or wettabilitie~, etc., or, perhap~, tha thr~Q lay~s woul~ use th- ~am- mono~r, e.g., YDM, with wh~ ch throe diff erent pro~ei~o or other llgand- wer~
immobilized to make; for 6xa~ple, a ~impl~-to-u~ immunodiagno~ti~
-d-~c~.
~., Ex~m~ 17 - Pr~arati~_o~ Corona-Trea~Q~ AzL~çtQn~-~un~tlonal Su~orts The corona depo~ition o~ ~umpl~- (pr~pared accord~ng ~o Example 16) wa~ carriod out in a bell~ar ~y~tem uaing two m tal.
roller~ ~10 cm dia~et-r, 15 cm long) for ~lectrodea. The grounded el~c~rod~ was co~rsd with a ~ ~m th~ck ~le~- of silicone rubber and ths el~ctrod~a wer~ ~sparated by a 1.7 m~ gap. The sampl~s w~re W O 93/25594 PC~r/US93/04Ci5 2 13~9~-, mounted on the eilicone ~leeve u~ing tape The roller~ rotat~d at 25 rpm causing the ~a~ple~ to be repeatedly expo~ed to th~ discharge in the region of clo~e~t proximity betw~en the two roller~ The belljar was vacuated to remove th- air atmo~phere and backfill~d with 100 mtorr VDM and Ho to a pre-~ur~ of 1 atm The ample- were expo-ed to a 250 W corona discharge ~40 kHz) for 3 minutes of rotation ~approximat~ly 30 actual xpo-ur- to the di~charge) Alt-rnativ ly, similar to that de-cribod in Example 16, the ample~ were ubject-d to a nitrogen gas corona treatmont (1 atm) of 250 W (40 kHz) for 15 of rotation (2 5 d of expo-ure) Thi- wa~ followed by VDM treat~ent a~ de~cribed above Th~e ~ampl~- are indicated in Table 13 as the N~VDM treatment Th~ protein binding exp riment- were performed id ntically to thoso de-cribed in Example 16 xcopt that the p~c1fic radioactivity of the Protein A wa~ 1300-1700 cpm/~g of prot-in ~abl~ 13 - The Cou~lins of Protein to Corona-Treated Material~
' Prot-in SDS Prot-in -~ ' 20 ~Binding ~ tanceCoupling M-t-rL~lTr-atment ~ ~ua//cnf) l~) Cl / SQ
, PE Control13 8/10 5 35/324 8l 3 3 ~ UDM 14 8/23 0 81l8612 0~19 8 2S N/VDM12 71 8 8 78~779 9/ 6 9 PP ~ Control1 2/ 0 4 15~150 2/ 0 1 : VDH ::1.9/ 1 0 58/401 1l 0 4 N/VDM~2 0/ 1 0 66/45,1 4/ 0 5 ~ PE~ ~- Control1 81 0 8 24/190 4/ 0 2 ,-^p~ VDM 1 9/ 0 9 491360 9/ 0 3 3~~ PSFE C,ontrol ~ 0 6/ 0 4 ~ 17/15 0 1/ 0 1 , VDH~ 1 5/ 0 8 70/571 0/ 0 5 " I , N/VDM~1 3/- 1 0 72/641 0/ 0 6 Corona~,tr--t~ent yiold~ r-~ult~ v~ry similar ~o tho-e ,~ ' ob--rv d in Exampl- 16 with pla-~a tr-atm nt Ther~ ar~ diff-r-nc-s ,in th ab,-olut- value- o~ om-,of th~ numb~r-, bu~ the g-n-ral - ~ aff~ct i- tho a~ addition of zlacton functionality to th~
f- ' ~-- 4S ~urf-c- ru~ult- in n incr~ n th amount of coupl-d prot-~n In th v nt~that ~t w-r- d -ir d to graft azLacton~-function~lity to ' r - lnt ior urf~c-- of a porou-, pr-r-x~-ting upport, on~ could hiold both -l-ctrod - w~th llicon r~bb r l--v - ~Lik- tbat -~ - d -crib d ~bov~) and mploy th- am- coron- di-charg- procodur~
SO u~ing h~lium ~- d cribod iA thi- Exumpl- to ~chi-v~ a p notrating VDM tr-~tm nt '~':
~ 36 ,' :, , ~ .

W0 93/25~94 213 L~; 01~ .~. PCr~US93/045~

~xam~le 18 - ~he Prep~ration o~ and Protein ~indina to Plasma VDM-- Treated Porou~ and--Fibrous Supstrates VDM was deposited onto the ~ollowing material~ in a ~low discharge procedure as de w ribed in Example 16:
S PP blown m~cro~iber web (basi~ weight of 60 g/m: and fiber ~iamete~ of 5-10 ~m) prepared by ~lt-blowing tochnigue~
disclosod in van Wente et al. ~Suporfine Thermopla~tic Fibers~
Indu~t~ En~ineerinq_Çhe~istr~, Yol. 48, pago~ 1342 et seq. ~1956) and ~an Wente ot al. ~Manu~acture of Sup~rSin~ Or~anic Fiber~, ~0 Report No. 4364 of Naval Re~earch Laboratorie~ ~May 25, 1954);
Celg~rdT~ microparous palypropylon~ ~embrane 2402 (commercially a~ailable from ~oechst-Celane~e, Charlotte, NC);
Polyurothane commerci~lly available from Dow Chemical under the tradenamo "Pell~thane 2363-65D~.
Rayon blown microfiber wob, such a~ tha~ u-~d in MicroporeTU tape, com~ercially availa~lo from Minne~ota ~ining and Manufacturing Company; and The PP film and hydrophilized poro~ PE ~e~br~ne tre~ted in the manner de~cribed in ~xamplc 16. She non-porou~ PP fllm wa~
al~o ~ubjocted to s~ultanoou~ troatment with a combination oS VDM
and hydroxyethyl methacrylate (HE~A) u~ing a glow di-eh~rgo (50 I, mtorr VDM, 10 mtorr HEMA, 15 W) to depo~it a 70 nm coatin~.
ProteLn binding experiment~ were ~erfo~med a~ described in Example 16, except that the blown ~icroSibor web and Celgard material- r¢guired O.2~ Tr~ton X-100 in order to be thoroughly wstt~d by th~ buffer ~olutionA.
Tho ~pecific radioactivitie~ varied fro~ 5100 to 6500 cpm~q of protein. Th- ~thanolamine qu~nching step- were for a total of 3 h. - -:

_ _ .

3~
J

W0 93/25~94 . Pcr/U~93/04~
2 1 3 6 0 ~ i ProtæinSDS Protein BindinqRe~i~tance Coupllng aterial Treatment lY9l5~ (uq/cm ) PP Control0.37/ o.i6 6/ 9 0.02/ 0.02 VDM 0.17/ 0.77 23/76 0.04/ 0.58 VDM/HEMAG.16/ 0.79 23t80 0.04/ 0.63 ~0 PE Control3.50/ 6.48 45/62 1.57/ 4.00 VD~ 1.7~/16.10 86/91 1o54/~4~71 R~yon Control0.06¦ 0.17 26/40 0.02/ 0.07 ~DM 0.08/ 1.33 34/91 0.03/ 1.22 PU . Control0.30/ 0.33 29/38 0.09/ 0.12 W M 0.19/ 0.68 47/64 0.09/ 0.44 PP/BMF~ Control0.031 0.77 13/15 0.004/0.04 VDM 0.03/ 3.91 25/93 0.009/3.65 Celgard* ~ontrol0.03/ 0.93 9/18 0.003/0.17 VDff 0.04/ 1~10 19/68 0.0~7/0.75 ~ indicated Triton X-100 in protein i~cuba~ion ~olution ~zlactone-modification of rayon ~a cellulo~e-~as~d ~y~the~ic polymer) and PU yi~ld 17-fold and ~o~r-fold prot~in eoupling increase~, re~pecti~ely. ~h- PP ~icrofi~er ~hows a tremendo~ hurldred-fold incr~a~e upon azlacton~ mod~fication. The modification of Celgard polyethyl~ne i~ ~sp~cially uE~ful becau~ i Celg~rd polyethylene i~ often ~ m~tQrial u~ed to mak~ microporous hollow fiber f iltration membrans~ . A~dition of ~ pl~otropic ag~nt ~uch a~ azlacto~e to hollow f i~er m~m~rane~ would incre~e their utllity many f old .

C~mnarico~9~w ~ischarcle ~eatme~t DOeB Not Penetr~te nto Poro~ ~terial Two 10 cm x 10 cm piec0~ of microporsuH PE me~bsane, 29 ~m thic~, prepar~d according to Example 23 of U S Pat No ~~40 - 4,539,256 (Shipman) wer~ tsp~d togother alons thair cdges and placed on ~he botto~ ~loctrod~ ~or pla~a glow discharg~ ~reat~ent in the bell~ar ~cuum ~y-tem according to ~x~mpl~ 16 ~h~ me~bran~ was trea~ed with a ~low di-charga of VD~ ~60 ~orr), a~ d~scribad in - ~xample 16 ~wo ~ampl~- w-r~ prepar~d, one w~th a 70 nm coating and ~S the othes w$th a 15~ nm coating of tho VDM-pla~ polym~r After tr~atmcnt, th~ upper lay-r o~ th~ two-layer con-truction wa~
eparatod from the lower layer, and analyzo~ by X-ray photo-lectron p~ctro-copy ~X~S) Both surfac~- of thi~ mQmbrana, th~ top ~urface ~xpo~d to the ~lectrical di~charg~3 and the ~ottom surface ~which wa~ in cont~ct with t~e lower ~ombrane), wero analy~cd W O 93J25594 2 1 3 ~ ~ Q -1 PC~r/U~3/045~

Sable lS - ~S_Analv~ of the External S~face~_o~_~ischarg~-Tr~ated 20 um ~orouff ~..nn~3nes Atomiç Ratios S ~ QL~
70 nm - top 0. 23 0.12 70 nm - bottom 0.0 0.0 150 nm - top O . 22 0 0 12 150 nm - bottom 0.O O.O

The ~op ~urf~cea clearly have a~lacton~-function~lity, a~ evidenced by tbe oxygen and nitrogen atom cont~nt. The bottom surface~ of the ~embranets are untreated PE, with no oxygtan or nitrogen pre~ent. Thi8 demon~tralt~d that the electrical-tlwharge-deposited palymer of VDM does ~ot penetrate appreciably into the p'OEei'B of the mem~rane, ~ven a very thin membrane and o~ren when very hea~ily loaded with VDM ~a~ ~vid~nced by the ratio~ of O and N to C
which are very near the theoretical value~ ~xpec~ed for an ~infinite~y thick~ layer of pure ~DM, 0.29 and ~.14, re~pectively).
Th1s cxp-ri~ent demon3trated the fea~ibili ty o~ the ~multilayered" ~.n~l~ membran~
de~ice~ discu~lHed in E~ample 16. ?
~x*m let3 20-Sl -- Cro~ ed ~zl~t~nç~un~tls~LL~Qa~ on Non~o~e~ pQlvmçrLc Su~QEt5 Sablo 16 ~alow ~how~ the r~ult~ of a s~rien of ; 30 exporiment~ to prepare cro~link~d azlacton~-func~onal coating~ on su~face~ o~ nonwovetn-poly~eric upports. Thta m~thod~ o f prepasaeion follow Tabl- 16.

, .
- .

. .

, _ _ WO 93/25~94 PCI/US93/0~
21~n~ ~i - ~able 16 - Azla~tone - Functio~al_Coat~
~ , ~ ,,.
l ~SDSCOUPLED
MONOMER FORMULATION ¦ NONWOVEN RES. PROTEIN A
l __ _ ~g/cm-. . ~ _ -~ ~ ~ . _ 20:20:60 EGDffA/VDM/HEMA REEMAY-2 93 21-17 ¦
21 (50g in 400mL ~PA) REEMAY-1 68 1.45 ¦
22 PET S8 86 11.04 j .
23 P~T LB 82 7.70 ¦
24 PP OE 53 1.77 ¦
1~ 25 PUR 74 3.02 26 . . COS$ON 89 20.53 27 80:20 T~P~MAJVDM OER~X 75 3.70 ¦
23 50g in 400mL Hexan~ PE~ CW 65 2.91 ¦
29 DUPON~ SONSARA 85 9.53 ¦
PE/PF-3 64 65.20 i 31 PEJPP-10 37 16.66 ¦
_ . ................. . ~ I
32 70:20:10 BAJVDM/TMPTMA DUPONT SON~ARA 68 1.43 3334 (50q in 400mL Hex~ne~) CFX NYLON 52 0 29 RAYON 73 2.06 36 _ pP NP 26 1.07 37 70:20:10 BA/VDM/EGDMA DUPONT SONTARA 81 2.71 : 38 (50g in 400mL Bexane~ CFX NY~ON 69 2.09 39 PP . 33 0.27 ~AYON 77 9.92 41 _ _ pP NP _ 4 0.23 42 70:20:10 IOA/VDM/EGDMA DUPONS SONSARA 66 1.38 : 43 (500g ~n 400m~ H~xane~) CFX NYLON 72 1.37 : 44 PP 36 0.21 ~: 30 45 R~YON 75 1.28 l ~- 46 PP NP 32 1.04 I
. , , , . , I
4~ 50:20:20:10 3~PONT SONTARA 74 1.~9 i _ - 48 IB~A/BMA/VDMf~MP$MA CFX NY~ON 63 1.27 49 ~50g in 400~2 H~xane~) PP 49 O.30 AAYON 76 2.49 51 . PP NP 34 2.37 _ ~_ :~ _ _, ~
~0 PROTES~ A protein A coupled using 1.5M ~ulfate in 0.2M ~odlum phocphate buffer REEMAY-l Style 2200 punbonded polye-ter from XEEMAY cf Old Hickory, Tonne~-e-RE~AY-2 S~yle 2295 punbonded polye~t-r from REEMAY
~5 ~UPONT SONTARA Rayon/poly~-t-r ~ba-i- w-ight 135 g/m2 from DuPont) C~REX Typ- 23 Nylon 66 (baJ~- weight 34 g/ml) from Flberweb o~ Simp-on~ille, S.C~
- - -- PET TB Th-rmal bonded polya~ter pr~p~red ~rom CE~80ND
-- br~nded b~oomponent fib-r ~2~ fiber dia~ter) on : - SO air laid w~b for~or from Rando ~achinQ and uaing aLr circulat~on o~-n for bLnding PET EB Air laid polye~ter b~nded with Rohm ~nd ~aa~ branded lat-x.
P~ OE Oriented, embo--~d polypropyl~ne ( a~ f~ber 5S ~iameter)melt blown according ~o U.S. Patent No.
4,988,560 (Meyer t al.) W O 93/25594 2 1 3 6 0 0 ~ P(~rfUS93/045~

PUR ~elt blown polyurethane ~8~ fi~er diameter) melt blown according to Wento et al. "Superfin~
- ThermoplastiC Fiber~ in ~8, -~ D~D~
Che~i~trv, Vol. 48, pAge 1342 e~ seq. (1956) S CO~TON Sounlac~d cotton from Vffratec Corporation PET C~ Mechanically laid, embo~ed poly~t~r ha~ing 2 denier fiber PE/PP-3 Needlepunched,.air laid polyethylene sheathed polyproyene having 3 denier fiber PE/PP-10 Needle punched, air laid polyethylen~ sheathed polypropyl~n~ having 10 denier ~iber CFS Nylon Melt blown CFX nylon copolymer from Alli~d PP NP N~edlepunched, ~ir laid polypropyl~n~ baving 205 g/m2 b~$~ w~ight PP Air laid polypropylene having 205 ~/m2 ba3i~ w~ight RAY9~ Neodlepunched, air laid rayon having 135 g/m~ baais waight Exam~les 20- 20:20:60 EGDMA/yDM~H~A Coatinq_Q~ S~unbonded Po~yeoter A spunbonded polyea~r ~ample ~E~NAY, ~tyle 2200;
15 cm ~quare) wa~ dippod into a 2-prop~nol ~olution of 20 pa~tE
EGDMA, 20 part~ VDM and 60 par~ HEM~ ~prepared by di~ol~ing 10.0g EGDMA, 10.0g V~M, 30.0g E2MA, and 1.0g Daroeur~ 1173 in 400~1 2~
. propanol), and hen pr~-ed be~we~n ~h~et- of polyQthyl~ne to remove exce~ ~olution. After purging th- ha~pl~ with N2 ~o~ 3 minute~, tho monomer coa~ing was polymeriz2d by expo~ure, undor N., to low int~n~ity W i~radiation for 12 minutos. The 8upport wa~ then ~oaked in 2-propanol for 1 minute and air ~ied. Analy~i~ by attenuat~d total reflectanoe IR ~ATIR~ rev~aled the characteri~tic ~zlactone carbonyl ab00rption at 1820 cm~ indicating azla~tone incorporat~on in the polymer ~oating. S~ ~naly~is of ~he tr~ated polyo~ter rQv~led no particulat~- and ind$cated h uniform ~oat$ng of the f i~er~. The azlactone-functional ~punbonded poly~ter ~upport couplRd 21.17~g of radiolab~led Protein A per cm ~m~a~ured after SDS treatment)~

Exam~le~ 21, 23-26 - . __ The nonwoven-~u~pl~s 21 and 23-26 were coated and cur~d in the ~am ~anner a~ Exumple 20 ~xcept a diffQrcnt nonwoven wa~ employed, a~ $dentified in Table 16.

~xam~le_~ - 20:2Q:60 EGDHA~Y~M~HEMa~Co~tlnq on Th~rmal Bonded Polve~te~_ Th- th-rmal bonded poly~ntQr ~mpl~ ~25 ~m thick, 15 om s~uarej wa~ coated-a~d~-cured ln th~ ~anner o ~xampl- 1.
~naly~iJ of the flhi~h~d-~upport by ATI~ r~vealed th~ ~zlactone carbonyl ab~orpt$on at 1820 C~'. SEM analy~i~ revealed the coating SO to ~e grainy and particulate. The the~mal bonded polyo-t~r azlac~one-func~ional support coupled 11.04 ~g of radiol~boled W O 93/~5~94 PC~r/USg3/045~
2 1 ~ J- ' Protein A per cm- ~measured after SDS treatment~

Examole 30 - 80 20 TMPTMA/VDM ~oatinc on Polv~roovlene A polypropylene sa~ple (3 den-ier, needle punched, 15 S cm ~guare) wa~ dipped into a h~xane solution of BO 20 ~MPT~A/VDM
(prepared ~y dis~olving 40 0g TMPY~A, lO Og VDM, and l Og Darocure 1173 in 400mL hoxans) Exce-- solution was removed from ~he wob by pre~eing it betw~en ~heet- of polyethylone Aftor pLr~ing with N2 for 3 minuts-, th- monomer coating wa- polymerized by oxpo~ure under N2, to low inten~ity UV irradiation for 13 minutes Becau~e of the thicknec- of this sample, the web was fl$pp~d over, purged, and irradiatod for an additional 6 minut~s Th~ azlactone-functional support wa- then rins~d wlth h-xan~ and air-dried SEM analy~i~ of th~ compo-ite rsv-al-d a grainy particulate coating with Bome agglom~rated particle~ The Daiwa compo~ite coupled 65 2 ~g of radLolabled Protein A per cm ~mea~ured after SDS treatment) .
ExamDles 27-29 and 31 Sh- nonwoven sample- of Exampleg 27-29 and 31 t15 cm guar-) w~re coat~d and cured in th~ manncr of Exa~ple 30, except that- Example- 27-29 wer~ irradiated on one side only for 6-11 min and ~xamplo 31 was irradiated on both ~ides in succesaion for 7 min each 1 25 Examole~ 32-3Ç

The nonwoven ~amples of Example~ 32-36 were dipped !~
~nto a h xan-- solution of 70 part- BA, 20 part~ VD~, and 10 part-of TMPT~ prcpar-d by dL-~olving 35 0 g BA, 10 0 q VDM, 5 0 g ~TMPTMA,~and~1 0 g Darocure 1173 photoinltiator in 400 ml of hexanes and then wor- pre-~ed botween heet~ of polyethyl~ne to remove exce-- olution Aft-r purging with N. for 3 min , tho monomer - - coat~ng was polymerized by expo-ure und~r N2, to low Lnten~ity uv - radiation for la min- Tb- azlactone-functional upport~ w~re ~oaked and rin~d with h~xane- and th n dri~d under N2 - -Examole- 37-46 Th- nonwoven ample- o Exampl-- 37-46 (10 cm 4~----~gu r-) wer- coated with a h x-n c olution of 70 part~ 8~, 20 part~
-~ -~ ~VDM, 10 part- ECDM~, contaLning 2t Darocurcn~ 1173 photoinitiator and then cur-d in the 0anner of Ex~mplec 32-36.

ExamDl~ 47-51 4S The nonwoven ample~ of ~xamp~ 47-51 (10 cm `:
W O 93/25594 ~ 1 3 6 0 ~ ~ PC~r/US93/045~5 square) were coated with a hexanes ~olution of 50 parts IB~A, 20 parts ~MA, 20 part~ VDM, 10 part~ 5MP~MA, containing 2~ DarocurerM
1173 and then cured with 12 minute~ o irradiation in the same manner a3 Example~ 32-36 Exam~le 52-- PreoaratiQn Qf AzLactone-FunctionaL
Polvm0thv~,methacr~late poly~methylmethacrylate) ~PMMA çommoreially a~ail~ble as Per~pex CQ, W from ICI) button~ were expos~d to on~ of ~evcral electron bea~ do~ages grafting at conditions of 10, 20, 30, 50, or 100 kGys di~charged at 100 kV ~n N.
gas a flow of les~ than 4 ~am/min, at a lin- peed of 9 2 ~/min, with O content les~ than 50 ppm The act~vated PMMA ~ub~trate wa~
~mmediately transferred into YD~ monomer for graftinq The grafting wa~ immediately tarted on the surface of the support ~he buttons were washed with VDM monomer and anhydrous ethyl-ether When the actlvated support wa~ er3ed longer (e g , ~eversl hour~), in VDM
monomer, th- VDM-grafted PM~A wa~ di-solved in the VDM monomer Th-refore, to ~inimi~e di-solution, a hept~ne/VDM (75/25 wt ~) olution wa- ubstituted for VDM monomer It was found that ~he surface of the button support remalned intact As a control, the PMMA buttons were expo~ied to electron b~a~ only to e0 how much polymeric chains wer~ ~egraded by electron beam Evldence of VDM
grafting on the PMMA button was confirmQd by AT~-FT~R, 13C NMR and XPS ~ESCA) The molecular weight distribution cur~e lndLcated there wa- no degradation oS the polymeric chaLn~ of tho ~upport at all under the condltionfi of 10 kGy- ~lectron beam doAe ~xamole 5 -- Azlactone-un~tional PMMA PreDared and ~eacted with bmine-TermLnated HeDarin PMMA casted film (P~rspex, CQ, W ; cO lmm thlckne~
wa~ ~xpoEie~ to the electron boam in a ~ blanket, and then conv~yed into th~ glo~e box under N2 g~- where the aotLvate~ f$1m wa-3S i~m r-~d $n VD~ monomer Sor twelve ho~r- at room temper~ture~ She fllm lo~it all of ~t- original h~p but wa- not di~i-olved in tho VDM
olution Th~ VDM monomer w~ d-canted and the grafted P~MA
di-~olv-d ln chloroform and preci~it~t-d into hexane ThL- top wa~
~op~atod two tim - to r-~ov- un~raft-d VDM monomer Finally, the - ~0 az~actone-functional PMMA upport wa- di--olved in ehloroform, c--ted onto an aluminum plat-,and~p~-lod ~way from th pl~t~ to form a thln film for analy-i~ Tranumi~ion IR p4ctrum of th- grafted PMMA ~how-d ther- wa- a trong peak of car~lonyl group of VDM at 1820 cm~ -~S

WO 93/25~94 PCI`/U~i93/04~5 213~

To prepare amine-ter~inate~ heparin, 1 g of heparin - (commercially available from Dio~ynth) wa~ di~sol~Qd in 300 mL of water. 10 mg Sodium Nitr~a WdB added to the 301utio~ and adjusted the pH to 2.7 with lN hydrochlo~ic acid. The ~olution wa~ adju~ted to 7.0 and dialyzed again~t 3 L of water in 3500 molecular weight cut-off dialy~i~ tu~ing. ~he solution was concentrated and lyophilized to produce heparin-aldehyde.
1 g o~ heparin-aldehyde was di~olved in 100 mL of th~ buffer solution ~1~ eitrate, 0.9~ ~odium chloride, pH ~ 6.5). 0.5 g of ammonium ~ulfate and 0.25 g of ~odium cy~noborohydride were ~dded to the olution ~nd ~tlrred for five hour- at room temperature. Th~
~olution wa~ dialyzed with 3500 mol~cu!ar weight cut-off dialysi~
tublng agA~nst water. The solution was concentrated and lyophLli~ed to produce am~ne-terminated hep~rin.
~hen, to react amLne-ter~inated hep~rin with azlactone-functional PMMA, the fi}~ prepAred acoording to thi~ example wa~
reac~ed with a solution of 0.25 9 of a~ine-terminated heparin which was dis~olved in 50 mL of buffer solution (pH - 8.8), and stirred for several hour~ at room temperature~ The heparin~zed-film was rin~d w~th water and dippod in 1~ ~olution of toluidine blue ~Sigma Che~ical) in w~tor for ~taining. The film ~tained ~ ~iolet co}or - within a few minute~ to ~how heparin wa~ attached to the f~lm.
.

~xamrie 54-- Azlactone-~ynctio~al ~ A Pre~red in aeDt~nç

PMMA ca~ted film (Per~pex C.Q./u~ from ICI; le~
than 0.1 mm thickne~) and a P~M~ button (2.75 mm thickne~a) were both expo6ed to electron beam (lOkGy- at 120 kV) in a N2 blanket and th~n conveyad in~o the glove box under N2 gas where the activated _ film and the a~tivated button wera ~ot~ immer~od ~n a mixture of heptane/VDM (75/25 wt.~) at room temperatur~ and 45 up to 15 hour3.
After qrafting, both form~ of the VDM-grafted PMMA wes~ wa~hed with anhydrous ethyl-~ther. AT~-F~IR pectra of the VDN-~rafted PM~A
Jhowei-th~rc wa~ a otrong peak of carbonyl group of YDM at 1820 cm'.
3~
E~am~lç 55-- Azlactone-~unccl~ y~ 9~ h Corona D~char~ç
~ ~- ~he corona-di~charg a~-~m~ly of ExampLe 17 above waa placcd ln a ~lov- box filled wLth N2 gas. ~MMA fil~- ~ro~ ICI
~0 and 1-3~ than 0.1 mm thickne~-) wer- xpo~ed ~o corona-disoharg- in -- -d~f~-r~nt condltion~ at 150 to 300 watt- for 0.4-4 ocond~ oxpo-ure ~ ~ diccharged at 62 kHz (at 150 watta for 0.4 and 4.0 ec~.~ at 200 w~tta for 1.2 and 4.0 ~eC~.t and at 300 watt~ or 1.2 and 4.0 ~ec~.
- Th activated PMMA film~ w re imm~r~-d in h~ptane/V2~ mixtura (75f25 4S wt.~) at room temperature for 2 hour~. Ev~dence of VDM grafting 21 ~,~0~
WO 93~25~94 PCI/US93/04555 wa~ ob~erved by ATR-FT~R ~n the~e instance~, there wa~ no evidenc~
of molecular weight degradatio~ From ~tudying thes- example~ 52-55 and the prior ex~mple~ form~ng biologically activo ~nd u~eful adduct supports, it i~ possible to convert a hydrophobic PM~ u3eful a~ an S intraocular len~ into a hydrophilic PMMA coated with an anticoagulant or othor biocompati~le hydrophilic and/or biologically active material by reaction of azlacton~-funct~onal moieties with an anticoagulant or another nucleophile-termin~ted hydrophilic moiety Exam~le 56-- DLs~er~ion Polvmerization of Azlactcne-~u~çtional Particle~ in Pol~ethvlene Membrone-Three microporou~ PE membranea prepared according to Example 23 of U S Patent 4,539,256 (Shipman) a~d one hydrophilized microporous PE me~brane prepared aecording to Example 22 of lnternational Patent US 91tO7686, ~ach having a ~ize of l5cm X t5cm, w~rc each placed on a slightly larger piece of den~e PE film and were saturated with a solution containing lOg of VDM; lOg of ethylene glycol dim~thacrylate ~EGDMA); 30 g of 2-- hydroxyethylmethacrylate ~HEMA); 1 g of photoinitiator (Darocure ll73 commercially avallabl- from E Merck), an~ 400 mL of i-opropyl alcohol (net olid- of ll l wt ~) Another piec~ of dsn-c PE film wa- then placed on top of th- aturated membrane followod by rubbing 2S the ~andwich~ con-tructlon to remov- any xce-- solution The andwich con-truction wa- plac- in the bottom of a N2 purged box ha~ing a Pyrex b.and glas~ top, and the ~andwich con~truction wa~
irradiated through th~ Pyr~x wLndow u~ing two fluore-ccnt W ~blackn lights having emi-sions at a max~mum of 360 nm ~he thre~ PE
amplo~ were 'rradiatod for 5 min, 10 min, and 15 min, re-pectively She hydrophilized PE -ampl- wa~ lrradiated for lO min Follow~ng irradiation, the andwich con-truction~ wero removed from the box, separated from the PS film ~he-t~ Except_for the PE ~ample irradiat-d for 10 min, wh$¢h wa8- dir-etly rinoed with isopropyl alcohol, each ~ampl- wa~ drl-d and th-n rin- d with i~opropyl alcohol for lO min Scanning lectron miero-copy ~SEM) of tho zurface and eros~ ction of ach umpl~ wa- performod ~xcept for th- hydrophil'zed P~ a~pl-, SE~ ob--r~ation of eros---oction-howod nu~h~ -~u- particl-- and particle elu-t-rz Ind'~idual particle~ wer- c~ n b tween 0 4 and 0 8 ~m in d~am ter ~ow-ver, ny particl-~ w r- al-o gqlosnr~t *-into a ma-- which wa- largb a~
S ~m in diam ter On the urfac~ of th -o ample- waJ a 2-5 ~m thick cake of agglomeratcd partlele- ha~ing average d~amet-r~ of ~kout 0 6 ~m, and on th urfac- of ~ a~ple- but th- PE ample rin-ed before drying, ther- wer- ar-a- of a den~- ~kin layer of W O 93/25S94 PC~r/US93/04~5 ~13~

about 1-2 ~m thick on the out~ide ~urface The PE ~ample rin~ed directly wa~ Rkin-free and had many particle- trapped within the void spacos throughout the membrane cro~ ectlon The hydrophilized PE ~ample was mo~tly ~kinnod and contained few if any particle- within the cro~r-~ection ~he interior of thi~ membrane appeared to hav~ a rough coating on the fibril ~urface~ Infrar~d peetroocopy of th- PE ~ample rin~ed diroctly howed ~trong ab~orbance- at 1822 cm~ and 1671 cm' (indioating VDM) and at 1728 cm ' (HEMA and EG~MA) which ~onfirmed the pre ence of cro~slinked VDM~HE~A copolymer Exam~le 5?~ ersion PolYmeri~tion of Azlactone-functional Particle~ in P~lvDro~vlene Membrane A microporou- PP membran- ~pr~par~d aceording to Sxample 9 of U S Pat No 4,726,989 ~Mrozin~ki)) wae treated in the ~ame manner a~ the hydrophilized PE membrane in Example 55 In thi~
in~tance, the ~amplo wa- ~k$n-fr-e and had many particle~ trapped within the void ~pac-~ throughout th~ membrane cro~-eection ~0 Exam~l- 57- Di~er~ion Polvmerization o Azlactone-functional Particle with a Stab~lizer $he proc~dur- of Example 55 wa~ repeated with the addLtion of 2 23 wt /vol ~ of poly~vinyl pyrrolidone~ (PVP R-30 commercially available from EM 8ei-n~--) to the monomer mixturo of Ex~mpl~ 55 and aturated into tho por-~ of a PE m~brane propared according to Ex~ple 23 of 4,539,256 (sh~pman) Aft-r irradiation, i~opropyI alcohol rin-inq wa- employed For compari-on, the ame PE
30 m mbr~ne wa~ aturated with tho mixturo of Exampl- 55 Without PVP
~tabiliiér,~ a bimodal dl~tribution of particlo- m~a-uring ~bout 0 15 ~m in diam~t~r were found within th por~r of the PE membrane with a f-w 2 0~~m d-i-um~t~r partlcle- par-ély ~cattered through th ample With tho PVP pr --nt $n th- monomer mixture, the partlcle~ w~re 3S aboùt 0 2-0 5 ~m w~th no oceurr-nc- of th- much larger partlcle~ I
Thus, PVP ~tabillz~r improv-- the particle i2e dlstributLon partlcl-- within~void p~ee- of the PE membran ~ExamDle~58-65-- Di-~er~ion PolYmerization w~th~ lteratlon of t~e Sus~ort: SQvond Vse~ulne--= _ , . .
PP ~mbrane- umplo- pr par d accordlng to Example 9 of U.S Pat. No. 4,~29,989 (Mrozln-ki) wer- u~d as ~h- outer layer~
in a thre- lay-r m-mbr~no tack, ha~lng a middle layor of a hydrophilized PE m mbrane pr-pared accordlng to Ex~mple 23 of U S

W093/25~;94 2l36~a~ PCr/US93/045 Pat. No. 4,539,256 (Shipman) and hydrophili~ed according to Example 22 of copendin~, ooa~ ned applic~tLon 07/775,969 (Gagnon et al.) ~he hydrophilized middle layer i~ denominated the H-PE layer. The membrane ~ta~k wa~ placed upon a piece of den~e LDPE film and ~aturated with a monomer solut~on of 0079 moleo VDM, 0.48 mole~
EDGMA, 0.25 moles HEMA, containing 2~ Darocure 1173 photoinitiator, dissolved in i~opropyl alcohol in tho amount of percent ~olids ~bown in Tables 11 and 18 b~low, according to their thccretlcal percent ollds. Examples 58-61 w~re the top layer of PP in each ~ample;
Examples 62-65 wers the middl~ layer, H-PE, in ~ach sample.
(Unmodified controls were al~o te~ted.) After saturation, another piece of den~ LDPE film was placed to eo~er the membrane ~tack,and the exce~s ~olution was ~queezed out with a rubber roller. The membrane ~tack ~nd the cover LDPE films wera ~hen irradiated for 20 minute~ with a b~nk of 4 fluoresc~nt ~black lightc~ having ~n emi-Yion ~aximum at 360 nm, under ambient temperatur~, pre~sure, and atmo~phere.
Sample~ of Examplo~ 58-65 were ubjected to BET
analysis (using the mathod de~cribed ~n Example 3 abo~ and pore ~ize analy~i~, Gurley an~ly~i~, porosity, and water permeability analy~i~ (using the methods described in PCT Pu~lication W0 92/07899 ~Gagnon et al.)~. The re~ults for ~urface araa ~re ~hown in ~able 17; the porou~ property result~ are hown in ~abl~ 18. Exumples 58-61 how that where di~tinct diffpcr~ion ~ead~ develop withln the por-~ of the m d rane, the urface ~rea i- ~ignificantly ~nhanced ~h~- nhance~ent i~ urpri~ingly obtained without a ignificant dscrea-e of oth-r microporou~ properties of th~ ~embranes, a~ een in Sable 18 - 8y contra6t, the re~ult~~o~ Example~ 62-65 ~how that a coating of eros~linked V~M-co-HE~A~ -forme~ in itu on the internal poro ~urfacc~ Thlz i~ hown by a decrea~e in specific area/frontal ~urface ~rea ratio, with th- exception being Exumple 65, whLch had a ~laey~ ne~work of cro~ nked di~per-ion b ad~
~llling the pore~ of th- middle layer H-P~ Also in contra~t to partLculate addition~, coating~ of Example~ 62-64 erved to decrsa-e ~omewhat the flow thsough propertie~ (~e Gurley and water permeability propertie~) of the membrane, although the pore Rize~
thickne--, and poro~ity valu~- wer~ not imilarly aff-cted boyond usefulne-- of the azlacton~-functional- ~upport ~0 ~

WO 93/255g4 PCr/US93/~45~5 213~9~ `
_ ~ U I ~ ~ '~ ~ I U~ 0 ~ ~
,~ ~ lll . ~ , I
l ~ ~ o ~ u O ~ ~ ~ O
r ~D O el ~ o u. ~ ~ ~
_ ~ ~ n ~ ~ ~ r , E~ _ ,~1 1~ ~ ~ ~r N ~'1 N ~ ~
¢_ I
_ ~
3 ~ _ oo~o~ o~ooo ~ ~ E o ~r ~ ~, O t~ ~ o O
C~ 111 -- O N ~ U~ N t` 0 a~ ¢I t`
E~ ~ ~ i ~
~ 3 ~ ~ I ~ ~ ,, ~ r~ N ~q ~1 1 o ooooo ooooo Z ~0 -- . ~ o ~ o o~ o ~ ,~C _ 8 ~
~! ~ 3 .: Q I ~ ~
O m O~ O~ ~ O ~ 'It N ~ ¢~
_ _ c~ In 1` a~ 1 ~ O
"E ~ N 1~ 1 ~ 4 CID
~ _~ 0 o_ I ~ 1 ~I N I N U~ ~
U~, _-. I
- _~ O
~ C- ~ ~ O ~ O ~
~ --- V ~ C~
-- ~-- ~ . ~1, _ .,,,, , ~ _~

WO 93/25594 2 1 ~ S ~ Q, PCIJUS93/04555 1~ I
.-~ V~ l U ~, I ~~ I O
6J 9 ~ ~ ~D ~
E _ ~ a ~o 2 r~ ~t ~ ~ t ~1 1 0 ^ ~ D O ~ U) ~ r ~ 8~ ~r I ~ O ~P ~ o ~ ,~
~ O~ t~ I~
! ~ I ~ o ~ o 0 ~ Itl I C~ 0 ~ ~ N ~0 0 0 , ~ ~ a~ ~. ,. h, ~
z 5 .~ ,,.
~n ~ ~ r ~; _ 0_ ooooo ooo l o ~--- . ...
EdC E ~ ~ ~ ~ D o ~o:~ a ~ ~ _ - ~b C I -- - --I a~ ~1 O ~ ~ ~ O 0 0 ~ ,0 , -- -~ t~l ~ O ~ ~ ~1 ~ O N U- r~ ~ . _ .- , _ _~

~t ~ ~t ~ O _tC . i L~ _ ~

WO 93/2S~;94 PCI-/US93/045.~5 ~13~0~1l Examples 63-65 were also sub~ected to testing for - protein coupling analy~i~. Th~ ~amples were te~ted u~ing radioactively labeled protein A, according to the proc~durQ~ of Example3 5, 6 and 10 abov~ by incubating overnight in radiolabeled Protein ~, bufsered with sither 1.5 M ~ulfate buffer (SO,) or 250 mM
phophate buffered saline (~S), both ~t pH 7.5. .Af~er guenching of unrea ted azlactone moi~ti~ with ethanolamin~ nnd rspeated rinfiing in buffer, scintlllation counting wa~ done to d~t~rmine initial binding le~els. The ~ample~ were then incubated fo~ 4 hour~ in 1.0%
~odium dotecyl~ulfate (SD5), followed by scintillation counting to det-rminQ the hmount of coupled protein remaini~g.
Table 19 hows the result . -_ WO 93/25~i94 2 1 3 ~ 1 PCr~US93/04sSS

r~ ~
~ ~ E , ~ ~j 1~ In ~ o rl O ~ u~ ~ ~r 'D ~ ~

.

~ '.D O ~ ~O O ~ r~
~ o~ o ~ al o i~ii _ N ~ ~ ~ U~ Ul ~

i~ii C ~- l .. , C ~j ~ 1`~ ~ i` 10 V '~I _ ~ ~ D U'l o ~ ~ ~ ~ ~o ~ ~ ~, ~ ~ r---~ iii ~ 8. 0 0 ~0 ~

~ O Q
~ ~ r...~ ~0.~
~U~ _ O t_ _, ~ o r~ ~ ~
~~ ~ ~ ~. ~ ~ ~ ~, ~ ~
~ 3 : N ~ ~ 1.~
lZ I 1~

~ii O O O O O O O O
i~;~ Y O ~ 0 0 O ~ 1~ 0 i~ 3 o ,~ ~n 5 o ~
_I_ -,8.
~i ' o o- ~ , ~ o~ 5~ o -~

LL~ ~--r~ `

W O 93/25594 PC~r/US93/04~
213~0~

These data show that the addi~ion of azlaotone functionality does çorrelate with an incr~ase in the percent of coupled protein, and that use of ~ulfate is pref~rr~d to ~se of saline as a buffer syat~m.
The ~xper~ment was repeated for s~ples of Example 63, except that the experiment~ w~ræ done a3 a function of the tLme allowed for initizl binding~ Tha amount of time ranged from 0.5 hours to 16 hours and resulted in init$al binding rangin~ from 21 ~g/cm~ for 0.5 hours to 48.9 ~g/cm2 for 15 hou~s.
~ho oxperiment was then r~p~ated for s~mples of Example 63, except that rather than incu~atlng in th~ prote~n A
solution, the protein A solution was flowed through th~ azlactone-functional membrane. In thia c~se, 3 ml of a 1 ~g/ml solution of non-radioactive, S0~-buff~red protein A solution at pH-7.5 was suctionod through a 25mm disk of th~ ~zlactone-f~nctional membrane using aspirator vacuum. ASter thrQe flow-through rins~ cycle3 with PBS bufSer ~olution and quenching of possibla unreacted a21actone moieti~- with 6 ml of ~ ethanolamin- ~buffered to pH-9 with 25 m~
pyrophosphate)~ the membrane uamplee w~r~ analyzod for Protein A
content u~ing the BC~ protocol publi~hçd by Piçrce Chemical Co- for 8CA Protein Assay Ro~gent (cat. No. ~3220/23225, Pieroe Chemical Co., Ro~kford, Ill.). It was found that the sem~r~ne~ i~iti~lly ~ound an averago of 22.3 ~g/ml o protein A u~ing the flow-through ~odo of binding, where the time of expo~ure to th~ protein solution ~ 25 wa~ 1BSA than about 3 ~inuto~. Thiz-Amount of init~al binding wa~con-~tont with initial bindinq lev-l~ for Example 63 u~ing a 0.5 hour incubation. $ho ad~antage o~ flow-through bindfnq i~ that binding is not li~itQd by th- rat- of dlffu~ion of protoin into pore~ o~ th- membrane. Flow-throuqh bindinq al00 de~on~trated that kinetic~ of ~nlt~al bind$n~ of protein to azlactone moietiea i~
appar-ntly ~ery rapid. ~~

Examoles 66-69-- _ Retai~ed Useful Porou~ Prooçrt~e3_0f Azla_~Q~e-~ction~1 Su~Q~Q ~ ~
Nembrane- were prep~rsd ~ccording to ~xampLe 3 above, using 50 kGy radiation and 10, lS, or 20 wtJ~ol ~ VDM
~olut~on~ a~ ted ~n Table 20 b-low. Although a ~igni~ieant a30unt of poly(VDM) wa- gr~fted to the membr~n~s, ~t i- apparcnt from ~ea-urement~ of-phy~cal propert~-~ that no ignificant change in the physical porou- properti - occurr-d. Moflt import~ntly, flow properti-- wero-not ~imi~shed beyond continu~d u-efuln -- of the ; ~zlactone-functiona~ mæmbran0s.

_ - 2l36n~

.1, ., ,~

-- 3 ^ 1~

lli~ I
1, ;~ _ ~
!

AMENDEO S~,E~T

WO 93/25~94 P~/US93/04~5 21;'~G~l , Th~ ~ampl~s of Examples 66-69 wers al~o teated for ability to couple protein A according to the method of Example 5 a~ove . The protein wa~ di~aolved in el~her ~ SO, or ~ P35 buf ~r . sy~tem as used in Example~ 63-65. Table 21 ~howa the re~ults below.

.

.
=

WO 93/255942 1 3 ~ O ~ L~ PCI/US93/04~5~

--' =~ 1`- --' ~
E ¦ r ~ a:~ ~D ~ ~ ~
s~ a ~ ~ O ~ o u ~;~ I ~ , o~ l I
I ~ ~r ~ ~ o~ u l I ~ t ~ 0 ~ , ~ I. . . . ~ , .
~ ~ C 'E ~ ~ u~ ~.o a~ ~ ~
~ O ~ ~ a~ ~ D 'O

:- ~0 ~

~C ~/ I----ii ~l ~ -- O ~ ~ N O . . . ~t t~ ~ _ O ~ o ~ ~D
- 2 :~ O
~ ~-- - - ~

~ -~ o ~ o ~ ~ ~ ~o o ~t cr~ ~o o ~r ~ ~ m ---~
~ O u ~D~O O ~ . . ~, ,, lY ~O o o O o o C O : - . , , I ~ ' . ~J
_I . _. N

: ~ ~ ~O$a~ _ o --- l ~ . .

W O 93/25594 P~r/US93/04~

2 1 3 ~

- The~ data~~how that the addition of azlactone functionali~y doe~
correlate with an increase in coupl~d protein. Also, the so~ bu~fer ~y~tem wa~ preferred.
Sample~ of Exampl~ 67 were al~o te~ed for r~te of binding during incubation binding and flow-through binding technique~ in the same manner a~ used for Exnmple 63 abova. For the incubation binding technique, the amount of ini~ihl binding ra~ged from 21.8 ~g/c~ for 0.5 hour~ incubation to 37.4 ~g/cn~ for 16 hour~
of incubation. For the flow-through binding technique, thQ ~mount of initial binding wa~ an dverag~ of 20.4 ~g/ml for a flow-through expo~ure of about 3 minute~. A~ with Example 63, flow-through binding technigues are pref-rred an~ al80 de~on~rate eontinued u-ofuLnes~ of the pre-exi~ting ~upport aftsr azlacton~-functionality i- added thereto.
Embodiment~ of the invention ar~ no~ limited by the above description and examples. For an ~ppreciation of the ~eope of the invent~on, the claim~ follow.

.

.
_ .

Claims (24)

What is claimed is:

1. A chemically reactive support, comprising: a porous pre-existing support having outer and interior surfaces and azlactone-functional moieties contacting only the outer and interior surfaces and modifying only reactivity of such surfaces while retaining useful porosity of the pre-existing support;
wherein said contacting is selected from the group consisting of chemically grafting the azlactone-functional moieties to the surfaces, crosslinking the azlactone-functional moieties in a coating over the surfaces, and forming crosslinked particles of the azlactone-functional moieties in contact with the surfaces.

2. An adduct support, comprising: a porous chemically reactive support of Claim 1 having azlactone-functionality only at outer and interior surfaces of the support and a liquid comprising a nucleophilic reagent reacted with the azlactone-functionality.

3. A method of preparing an azlactone-functional support of Claim 1, comprising the steps of:
(a) exposing outer and interior surfaces of a porous pre-existing support with high energy radiation to generate free radical reaction sites only on the surfaces and (b) causing azlactone-functional moieties to react with the free radical reaction sites to modify chemical reactivity of the pre-existing support.

4. A method of preparing an azlactone-functional support of Claim 1, comprising:
(a) covering outer and interior surfaces of a porous pre-existing support with azlactone-functional monomers, crosslinking monomers, and optionally co-monomers, (b) copolymerizing the monomers to form a crosslinked, polymerized, azlactone-functional moieties only at surfaces of the pre-existing support to modify chemical reactivity of the pre-existing support.

5. The chemically reactive support according to Claim 1, the adduct support according to Claim 2, or the methods according to Claims 3 or 4, wherein the azlactone-functional moieties comprise monomers, prepolymers, oligomers, or polymers comprising oxazolinone moieties of the formula:

wherein R1 and R2 independently can be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon atoms and 0 to 3 S, N, and nonperoxidic O
heteroatoms, or R1 and R2 taken together with the carbon to which they are joined can form a carbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or 1.

6. The chemically rective support according to Claim 5, the adduct support according to Claim 5, or the methods according to Claim 5, wherein the azlactone-functional moieties are derived from 2-alkenyl azlactones comprising:
2-ethenyl-1,3-oxazolin-5-one, 2-ethenyl-4-methyl-1,3-oxazolin-5-one;
2-isopropanyl-1,3-oxazolin-5-one, 2-isopropenyl-4-methyl-1,3-oxazolin-5-one, 2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one, 2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, 2-ethenyl-4-methyl-4-ethyl-1,3-oxaolin-5-one, 2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one, 2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one, 2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one, 2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one, 2-isopropenyl-4,4-pentamethylene-1,3-oxasolin-5-one, 2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one, 2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one, 2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one, 2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one, 2-isopropenyl-4-methyl-4-benzyl-1,3-oxasolin-5-one, 2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one, 2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one, 2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, or combinations thereof.

7. The chemically reactive support according to Claim 1, the adduct support according to Claim 2, or the methods according to Claim 3 or 4, wherein the pre-existing support is a ceramic, glassy, metallic, or polymeric material.

8. The chemically reactive support according to Claim 1, the adduct support according to Claim 2, or the methods according to Claims 3 or 4, wherein the pre-existing support is a porous, polymeric material comprising a woven web, a nonwoven web, a microporous fiber, or a microporous membrane.

9. The chemically reactive support according to Claim 1, the adduct support according to Claim 2, or the methods according to Claims 3 or 4, wherein the porous, polymeric material is a polyolefin and wherein the azlactone-functional moieties are derived from 2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one.

10. The chemically reactive support according to Claim 1 or the adduct support according to Claim 2, wherein the azlactone-functional moieties are grafted to surfaces of the pre-existing support which is a porous, polymeric material.

11. The chemically reactive support according to Claim 1 or the adduct support according to Claim 2, wherein the azlactone-functional moieties are crosslinked in a coating over surfaces of the pre-existing support which is a porous, polymeric material.

12. The chemically reactive support according to Claim 1, or the adduct support according to Claim 2, wherein the azlactone-functional moieties are crosslinked particles contacting the surfaces of the pre-existing support which is a porous, polymeric material.

13. The method according to Claim 3, further comprising comonomers are to copolymerize with azlactone-functional moieties at surfaces of the pre-existing support.

14. The method according to Claim 3, wherein the high energy radiation penetrates the pre-existing support to generate free radical reaction sites at surface within the pre-existing support and wherein azlactone-functional moieties react with said free radical reaction sites.

15. The method according to Claim 14, wherein the high energy radiation comprises uv radiation and the method further comprises adding a photoinitiator prior to the causing step.

16. The method according to Claim 14, wherein the method further comprises generating hydroperoxide functionality on the surfaces of the pre-existing support prior to the exposing step, and wherein the high energy radiation comprises heat.

17. The method according to Claim 3, wherein the high energy radiation comprise plasma discharge or corona discharge; wherein plasma discharge generates free radical reaction sites at outer surfaces of the pre-existing support; and wherein corona discharge generates free radical reaction sites at outer surfaces using dielectric protection over a single electrode or at interior and outer surfaces using dielectric protection over each electrode and using a helium atmosphere.

18. The method according to Claim 4, wherein the crosslinking monomers comprise ethylene glycol dimethylacrylate, trimethylolpropane trimethacrylate, methylenebisacrylamide, and divinylbenzene.

19. The method according to Claim 8, wherein the crosslinked, polymerized azlactone-functional moieties comprise a coating on the surfaces of the pre-existing support.

20. The method according to Claim 8, wherein the crosslinked, polymerized azlactone functional moieties comprise particles contacting surfaces of the pre-existing support.

21. Th adduct support according to Claim 2, wherein the nucleophilic reagent comprises biologically active materials, acids, bases, chelators, hydrophiles, lipophiles, hydrophobes, zwitterions, detergents, or combinations thereof.

22. The adduct support according to Claim 21, wherein biologically active material comprises substances which are biologically, immunochemically, physiologically, or pharmaceutically active and wherein the biologically active material comprises proteins, peptides, polypeptides, antibodies, antigenic substancee, enzymes, cofactors, inhibitors, lectins, hormones, receptors, coagulation factors, amino acids, histones, vitamins, drugs, cell surface markers, substances which interact with them, or combinations thereof.

23. The adduct support according to Claim 21, wherein the adduct support is an adsorbant, complexing agent, catalyst, or chromatographic article.

24. The adduct support according to Claim 21, wherein the adduct support is a mammalian body implant and wherein the nucleophilic reagent is an anticoagulant.