CA2190731A1 - Polymeric microbeads and method of preparation - Google Patents

Abstract

The present invention relates to porous crosslinked polymeric microbeads having cavities joined by pores wherein at least some of the cavities at the interior of each microbead communicate with the surface of the microbead. The present invention also relates to a process for producing a porous, crosslinked polymeric microbead as well as the product of this process. This process involves combining an oil phase with an aqueous discontinuous phase to form an emulsion adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets, and polymerizing the emulsion droplets to form microbeads. At least 10 % of the microbeads produced in accordance with the present invention are substantially spherical or substantially ellipsoidal or a combination of the two.

Description

~Wo gsl33553 F~~ C~ 79 2 ! 9073 1 POLYMERIC MIt~RnR~An.q AND METHOD OF PREPAR~TION
B~CKGROUND QF THE~ INVF.NTION
5 Field of the I~vention The present invention relate~ to microbeads of a cros61i~ked porous polymeric material and methods for preparing such microbeads. In particular, this invention is directed to a polymeric microbead of 10 exceptionally high porosity.
Descri~tiQn of the Prior Art Crns~l ;nkod, homogeneous, porous polymeric materials are disclosed in U.S. Patent No. 4,522,953 (Barby et al., issued June 11, 1985). The disclosed polymeric materials are produced by polymerization of water-in-oil emulsions having a relatively high ratio of water to oil. These emulsions are termed "high internal phase: ~;r,n~ and are known in the art as 20 "HIPEs". HIPEs comprise an oil c~nt;n-~rus phase ;n,lll~;nr~ a monomer and a crr~s~l;nk;ng agent and an a~ueous disrrnt;n~ us phase. Such emulsions are prepared by subjecting the combined oil and water phases to agitation in the presence of an emulsifier.
25 Polymers are produced from the resultant emulsion by heating. The polymers are then washed to remove any unpolymerized monomer/cros81; nk~r The disclosed porous polymers have rigid structures rrnt~;n;n~ cavities interconnected by pores 30 in the cavity walls. By choosing cl~L~Liate component and process conditions, HIPE polymers with void volumes of 80% or more can be achieved. These materials thus have a very high capacity for ~hsrrh;n3 and r~tA;n;nrJ
li~Iuids .

wo gsl33sS3 2 1 q ~ 7 3 1 P~ C~79 ~
Various modif ications of HIPE polymers have been described. For instance, U.S. Patent No. 4,536,521 (Haq, issued August 20, 1985) discloses that HIPE
polymers can be sulfonated to produce a sulfonated 5 polymeric material that exhibits a high capacity for absorption of ionic solutions. Other functinn~li7ed HIPE polymers prepared by a similar process have been disclosed in U.S. Patent Nos. 4,611,014 (Jomes et al., issued September 9, 1986) and 4,612,334 (Jones et al., September 16, 1986).
Although the existence of polymerizable HIPEs is known, the preparation of useful ~IPE polymers is not without its dif f iculties . Because the emulsions used to produce these polymers have a high ratio of water to oil, the emulsions tend to be unstable. Selection of the appropriate monomer/crnRsl lnkPr rnn--Pntration, 1 R; 'F; Pr and pmlll R; r; f~r concentration, temperature, and agitation conditions are all important to forming a stable 1 Rinn . Slight changes in any of these variables can cause the emulsion to "break" or separate into distinct oil and water phases. Furthermore, : llR;nn ~ n.ontR and process conditions that produce a stable prmll R; nn may not always yield HIPE polymers that are useful for their intPn-lPd purpose.
In addition to these problems, the costs associated with scaling up pro~ t; nn of HIPE polymer8 have prevented commercial development of HIPE polymer-based products. Proce8ses for large-scale production of HIPE polymers are known . For instance, U . S . Patent No. 5,149,720 (DesMarais et al., issued September 22, 1992) discloses a cnnt;n1ln~lR process for preparing HIPEs that are suitable f or polymerization into absorbent polymers. In addition, a method that facilitates such continl~nus processes by reducing the curing time of r ~~ ~ in a HIPE is set forth in U.S.
Patent No. 5, 252, 619 (Brownscombe et al ., issued ~Wo 9s/33553 ~ PCT/US95/068?9 October 12, 1993). Large-scale production of HIPE
polymers by such known processes, however, has been hampered by the lack of a cost-efficient means of removing the unpolymerized emulsion cnmr~-n nts from the 5 polymers.
All prior art processes for making XIPE polymers produce a block of polymeric material the size and shape of the vessel used for polymerization. The problem with producing HIPE polymers in block form is that it is very difficult to wash unpolymerized 1l R; .-n components out of a block of low density, highly absorbent material. The attempted solution to this problem has been to grind the blocks into particles, but this approach is unsatisfactory because both the drying and milling processes are costly, and there is a limit to the size of the particles produced by milling. For many applications, the removal of residual emul--ion ~ -ntc i- essential. Yet, to date, no cost-~f;r;-nt method for performing this wash step has been developed.
An additional problem with prior art HIPE
polymeric blocks is that the blocks have a skin that forms at the interface between the HIPE and the ~,-,ntA;n~.r used for polymerization. (U.S.
Patent No. 4,522,953, Barby et al., issued June 11, 1985, at column 4, lines 1-6). To produce a permeable block, and hence, to produce a useful product, the skin must be removed. Ideally, one would like to be able to produce a polymeric material having the desirable 3 0 characteristics of HIPE polymers but lacking the skin .
SU~M-RY ~JF THE lN VJ~ N
The present invention includes a material (hereinafter "microbeads") wherein at least about 10~6 of the material is in the form of substAnt;Ally spherical and/or substAnt;Ally ellipsoidal beads.

_ _ _ _ _ .. ..... _ .. . _ . .. . . . . .

W09sl33553 ~1q~l37 ~ 79 These microbeads have a porous, crosslinked, polymeric structure, char~tpr; 7~ by cavities joined by interconnecting pores. At least some of the cavities at the interior of each microbead communicate with the 5surf ace of the microbead .
The present invention also includes a process for producing a porous, crosslinked polymeric microbead as well as the product of this process . The f irst step of ~ -this process is to combine an oil c~nt in~ R phase 10(hereinafter an "oil phase~') with an aqueous discf~nt;nllous phase to form an emulsion. The oil phase of the Pml1lR;.~n includes a subst~nt;~lly water-insoluble, monofunctional monomer, a gubst~nt;;~lly water-insoluble, polyfunctional crosslinking agent, and 15an emulsifier that is suitable for forming a stable water-in-oil emulsion. The second step of the process is to add the . 1 R; on to an aqueou8 suspension medium to form an oil-in-water SllRpPnR; on of dispersed 1 RiC)n dropletg . The f inal 8tep of this process is ZOpolymerizing the Prmll R; ~n droplets to form microbeads .
In one ' i; - t, a polymerization initiator is present in both the aqueous dis~ (~nt; nll~ R phase of the ~IPE and the aqueous sllRrl~nR; on medium. A
polymerization initiator can optionally be included in 25the oil phase as well. In a variation of ~his f,mho~ , the oil phase; n~ "c styrene as the monomer, divinylhPn7Pno as the cro~sl;nk;n~ agent, and sorbitan monooleate as the . ~lRi~;pr~ l;t;nn, the oil phase ~,,nt~;nc the oil-soluble polymerization 30initiator azr~; Rnh; Rhutyronitrile as well as dodecane, which promotes the f ormation of interconnecting pores .
The aqueous r~;Rr~nt;nllOU8 phase includes the water-soluble polymerization initiator potassium persulfate.
The aqueous suspension medium includeg a 811RpPn~; n~
35agent comprising modif ied silica and gelatin as well as potassium persulfate. In another embodiment, ~Wo95/335~3 ~ 3~ 1`qo73~ F~ c~!~e79 polymerization initiator is present only in the oil phase .
The present invention also on, ~8~ microbeads that have been modif ied f or use in particular 5 applications. In particular, the present invention includes microbeads functionalized for absorption of liquids; carboni~erous structures produced from microbeads and a process for producing such structures;
and microbeads having a yel or pre-gel within the lO microbead cavities as well as a process for producing such micr~h~
In addition, the present invention includes the use of microbeads in a variety of applications including the use of microbeads as a substrate in 15 separation and synthetic methods; the use of microbeads as a substrate for; '-i1; 7;n~ a molecule such as a polypeptide or an oligonucleotide; and the use of microbeads in cell culture methods.
20 DET~TT.Rn DESCRIPTION OF TUR IN~IENTION
The Microb~q The present invention includes a cross1 inkl~d porous polymeric material, termed "microbeads ", wherein at least about lO~ of the microbeads are subst;~nt;;.~1y 25 spherical and or subst~n~;~11y ellipsoidal. The present invention also ; n~ a process ~or making such a material. A microbead is typically produced by cnlRp~n~i~n polymerization of a high ;ntorn~1 phase emulsion, termed a "HIPE". The microbead oi- t~e 30 present invention thus has many of the desirable physical characteristics of prior art HIPE polymers (such as those disclosed in U.S. Patent No. 4,522,953, Barby et al., issued June ll, 1985, which is incorporated by reference herein in its entirety).
35 Specifically, the microbead has a very low density due to the presence of cavities joined by interconnecting Wo95/33553 ~ ? t,~073 1 P~ , ''0679 pores. The bulk density of a batch of microbead8 in accordance with the present invention is typically less than about O . 2 gm/ml . The void volume of the microbead is high, preferably at least about 7096 and, more 5 pre~erably at leas~ about 90%. This high porosity gives the microbead exceptional absorbency.
Furthermore, because the intercnnnrct~lnPRR of the cavities in the microbead allows liquids to f low through the microbead, the microbead provides an 10 ~rr~ nt subgtrate for use in biotechnology applications such as, for example, CilL trg~raphic separation of proteins and peptide synthesis.
The average diameter of the microbead typically ranges from about 5 ILm to about 5 mm. Preferred 15 average diameters range from about 50 ~m to about 500 ~Lm. This 6mall size facilitates efficient washing of the microbead to remove residual unpolymerized lRinn ^nt~. Also, the process of the present invention can be used to produce microbeads of a 20 relatively uniform size and shape, which allows the wash conditions to be optimized to ensure that each microbead in a batch has been thoroughly washed. Thus, the microbead, unlike the prior art XIPE blocks, can be washed with relative ease . This f eature of the present 25 invention facilitates cost-F~ff;ri~nt scale-up of HIPE
polymer productio~.
An additional feature of the microbead is that the microbead is llskinless" such that some interior cavities and pores communicate with the surface of the 30 microbead. Thus, the microbead offers the advantage over prior art HIPE polymers that a porous polymeric material can be rro~llred directly upon polymerization, obviating the need for a skin-removal step.
The high porosity of the microbead renders it 35 useful as an absorbent material and also as a solid support in a variety of biotechnology applications, ~Wo gs/33ss3 ; 2 ~ 9 ~ 7 3 1 ~ 79 including chromatographic separations, solid phase synthesis, i -h;1;7~t1r~n of ~ntihofl;es or enzymes, and cell culture. Moreover, many of the physical characteristics of the microbead, such as void volume 5 and cavity size, are controllable. Therefore, different types of microbeads, spe~ d for :~
different uses, can be produced. A description of the general process f or producing the microbead i9 presented below, followed by a discussion of modifications for producing spec;~l;7ed microbeads.
Def; n; tions The term "microbeads" refers to a crosslinked porous polymeric material wherein at least about 10% of this material consists of substantially spherical and/or substAnt;~11y ellipsoidal beads. Preferably at least about 20% and more preferably at least about 50 of this --t~r;~l consists of subst~nt;~l ly spherical and/or subst A nt;;~11y f~ n; dal beads.
As applied to the r~, ~ of a ~IIPE, the phrase "subs~nt;~lly water-insoluble" ;n-l;c~t~ that any component present in the aqueous phase is present at such a low ~ ~n~-ent~ation that polymerization of a~ueous monomer is less than about 5 weight percent of polymerizable monomer.
As used herein, the term "bulk density" refers to the value obtained by dividing the weight of a known volume of microbeads by that volume.
As used herein, the term "void volume~ refers to the volume of a microbead that does not comprise polymeric material. In other words, the void volume of a microbead comprises the total volume of the cavities.
Void volume is expressed either as a percentage of the total microbead volume or as a volume per gram of microbead material (cc/gm).

wo gs/335s3 ~ ; 2` ~1 ~ 0 7 3 ~ 5 0~79 ~
A8 used herein, the term "cavity size", refers to the average ~1;: t~r of the cavities present in a microbead .
A8 used hereir, the term "porogen~' refers to an 5 organic compound that, when included in the oil phase of a HIPE, promotes the formation of poreg ~ ,nn--t;n~
the cavities in a microbead.
The abbreviation "DVB" re~ers to ~divinylh-n~-n~"; the abbreviation "AIBN" refers to 10 "azoisobisbutyronitrile"; and the abbreviation "PVA"
refers to "poly(vinyl alcohol) ", which is produced by hydrolysis of poly(vinyl acetate).
~licrobead Production The microbeads of the present invention are conveniently produced from a HIPE, which comprises an emulsion of an aqueous ~ c~nt; n~ phase in an oil phaOe. Once formed, the HIPE is added to an aqueous suspension medium to form a suspension of HIPE
20 microdroplets in the suspension medium. Polymerization then converts the li~uid HIPE microdroplets to solid microbeads .
In one: ' - '; , a polymerization initiator is pre8ent in both the a~ueous disc^nt; nll~llr. phase of the 25 HIPE and the aqueous suspension medium. A
polymerization initiator can optionally be included in the oil pha8e as well. In another ~
polymerization initiator is present only in the oil pha~-e .
~icrobead P~oduction Usinq an AqueouO Phase Polvmerization Ir;f tiator C -n~ of the Eigh Tnternal Phase ~3mulsion The relative amounts of the two HIPE phases are, 35 among other parameters, important determinants of the phy8ical properties of the microbead. In particular, 95/333~3 ~ ?~l~q073 ~ r~ ,3~!~e79 the percentage of the aqueous discontinuous phase affects void volume, density, cavity size, and surface area ~or the emulsions used to produce preferred microbeads, the percentage of aqueous disc~nt;n~ us phase is generally in the range of about 70% to about 98%, more preferably about 75~ to about 959~, and most pref erably about 8 0 ~ to about 9 0 % .
The oil phase of the emulsion comprises a monomer, a crosslinking agent, and an emulsifier that is suitable for forming a stable water-in-oil 1 Ri(-~r .
The monomer ^nt does not differ from that of prior art HIPE polymers and can be any subst;-nt'~lly water-insoluble, monofunctional monomer. In one embodiment, the monomer type is a styrene-based monomer, such as styrene, 4-methylstyrene, 4-ethylstyrene, chloromethylstyrene, 4-t-BOC-lly lLo~ye~LyL~ e. The monomer: , ^nt can be a single monomer type or a mixture of types. The monomer . ~nPnt is typically present in a concentration of about 5~ to about 9096 by weight of the oil phase. The concentration of the monomer c~r~nPnt is preferably about 15~ to about 509~ of the oil phase, more preferably, about 16% to about 38~.
The crosslinking agent can be selected from a wide variety of subst~nti~l ly water-insoluble, polyfunctional - ~. Preferably, the cro-cs~ ink;ng agent is difunctional. Suitable cross-linking agents do not differ from those of the prior art and include divinyl aromatic compounds, such as divinylbenzene (DVB). Other types of cross-linking agents, such as di- or triacrylic ~ 1R and triallyl isocyanurate, can also be employed. The crosslinking agent can be a single crnCRl ' nkPr type or a mixture of types . The crosslinking agent is generally present in a c~7n(~Pntration of about 1~ to about 90~ by weight of the oil phase. Preferably, the rr~nr~ntration of the _g_ .. . . , . , _, _ _ _ _ Wo ss/33s53 2 1 q ~J 7 3 ~ r~l,s~ - ~.,9 ~
cro.Al ;nking agent is about 15~ to about 50~ of the oil phase. More preferably, the concentration i8 about 1696 to about 3 8 96 .
In addition to a monomer and a crosslinking agent, 5 the oil phase co~prises an emulsif ier that promotes the formation of a stable emulsion. The emulsifier can be any nonionic, cationic, anionic, or amphoteric emulsifier or ~ n~t;~A~n of emulsifiers that promotes the formation of a stable emulsion. Suitable lc;f;ers do not differ from those of the prior art and include sorbitan fatty acid esters, polyglycerol fatty acid esters, and polyoxyethylene fatty acids and esters . In one ' ,~1; t, the ~ ; f i ~or is sorbitan monooleate (sold as SPAN 80). The ~ml~lA;f;er i9 generally present at a ~Ann~AAntration of about 4~6 to about 5096 by weight of the oil pha_e. Preferably, the rnn~Aontration of the Pmll1~;f;F~r is about 10~ to about 259~ of the oil phase. More preferably, the ~A~nn~A_ntration i9 about 15~6 to about 20~6.
In a variation of a first embodiment, the oil phase also r~A,ntA;n~ an oil-soluble polymerization initiator and a porogen. The initiator can be any oil-soluble initiator that permits the formation of a stable emulsion, such as an azo initiator. A preferred initiator i9 azo; -nh;YI -II yr~litrile ~AIBN) . The initiator can be present in a cnnc~ntration of up to about 5 weight percent of total polymerizable monomer (monomer ~ Ant plug crnAs1 ;nk;nj agent) in the oil phase. The ~Ann~AAntration of the initiator i9 preferably about 0.5 to about 1.5 weight percent of total polymerizable monomer, more preferably, about 1.2 weight percent.
The porogen of the present invention can be any nonpolymerizing organic ~ r-i or ;nAt;nn of compounds that permits the formation of a stable emulsion, provided that the ~ ulld i9 a good solvent ~WO95/33553 ~ q~ 073~ ,9 for the monomers employed, but a poor solvent for the polymer produced. Suitable porogens include ~n~Pr:3np, toluene, cyclohexanol, n-heptane, isooctane, and petroleum ether. A preferred porogen is dodecane. The porogen is generally present in a cnnrPntration of :--about 10 to about 60 weight percent of the oil phase.
The porogen cnnrPntration affects the size and number of pores connecting the cavities in the microbead.
Specifically, increasing the porogen r~nrPntration increases the size and number of interconnecting pores;
while decreasing the porogen Qnrpntration decreases the size and number of pores. Preferably, the porogen rnnrPntration is about 25 to about 40 weight percent of the oil phase. More preferably, the rr~rpntration is about 30 to about 35 weight percent.
In the fir8t: -,1; , the aqueous ~;qcnnt;n~lnus phase of a HIPE generally comprises a water-soluble polymerization i~itiator. The initiator can be any suitable water-soluble initiator. Such initiators are well known and include peroxide ~- ~q such as sodium, potassium, and ammonium persulfates; sodium peracetate; sodium peL~ c.Ll,ul,ate and the like. A
preferred initiator is potassium persulfate. The initiator can be present in a cnnrPntration of up to about 5 weight percent of the aqueous t1;qrr~nt;nllnu8 phase. Preferably, the cnnrPntration of the initiator is about 0 . 5 to about 2 weight percent of the aqueous disrn~t; n~ 1q phase .
~ nnPntS of the A~ueous Suspellsion l!lediulll After formation of a HIPE: by a process described in greater detail below, the HIPE is added to an aqueous suspension medium to form an oil-in-water suspension. The aqueous suspension medium comprises a suspending agent and, in the first ~ ;r-nt, a water-soluble polymerization initiator. The suspending agent wo 95/335s3 ~ 2 ~ 9 0 7 3 1 PCT/US9~/06879 can be any agent or combination of agents that promotes the formation of a stable suspension of HIPE
microdroplets. Typical droplet stabilizers for oil-in-water suspensions include water- soluble polymers such 5 as gelatin, natural gums, cellulose, and cellulose derivatives (e.g., hydroxyethylcellulose) polyvinylpyrrolidone and poly (vinyl alcohol) (PVA) .
PVA suitable for use as the suspending agent is produced by partial (85-929~) hydrolysis of polyvinyl 10 acetate. Also used are finely-divided, water-insoluble inorganic solids, ~uch as clay, silica, alumina, and zirconia. Two or more different s~ p~n~l;n~ agents can be combined. Indeed, in one ~mhorl;r^nt, the suspending agent is a, '-;n~t;nn of gelatin or PVA (88~
15 hydrolysis) and ~ l clay or silica particles.
Modified inorganic solid particles are produced by treating the particles with an agent that increases the hydrophobicity of the particles, thus improving the ability of the particles to stabilize the suspension.
20 In one ~mhnA; t, the inorganic golid particles are modified by treating them with a surfactant, such as asphaltene, in the presence of a suitable organic solvent. Suitable organic solvents include toluene, heptane, and a mixture of the two. The relative 25 nnnl ~ntrationg of the inorganic solid and asphaltene can be varied to produce modified inorganic solids of varying hydrophobicities. One measure of particle hydrophobicity is "contact angle", which reflects how f ar a particle penetrates into a water phase at an oil -30 water interface. =~ contact angle of 90 indicates thatthe particle resides half in the oil phase and half in the water phase. 1~ contact angle of less than 90 indicates that the particle penetrates further into the water phase, i.e., is more hydrophilic. In the present 35 invention, the hydrophobicity of the inorganic 801id particles is adjusted 80 that the particles promote the ~wo sS/33553 1 2 l q ~ ~ 3 1 P~ 79 formation of a stable suspension. In a preferred variation of this embodiment, the contact angle of the modified inorganic solid particles is about 65.
The suspending agent can be present in the aqueouq 5 suspension medium in aIly c~n~ntration that promotes the formation of a stable suspension, typically about 0.1 to about 10 weight percent of the aqueous suspension medium. For a preferred combination of suspending agents, a stable su~pension is obtained with 10 a PVA ~-,,nl-Pntration of about 0 . 5~6 to about 5~ and a inorganic solid concentration of about 0 . 05 to about 0.3~ by weight of the aqueous suspension medium.
In addition to a suspending agent, the aqueous suspension medium cont~;nR a water-soluble 15 polymerization initiator in the f irst embodiment of the present invention. The presence of an in;ti~tnr in the ~llcpPnq; ~n medium, as well as in the HIPE
microdroplets, accelerates the polymerization reaction.
Generally, rapid polymerization is desirable because of 20 the tendency of the sllqpPn~ion to break down over time.
The initiator can be any suitable water-soluble initiator such as those described above for the aqueous disront i nll~us phase of the HIPE . In a preferred variation of this ~rnho~1 t, the initiator is 25 potassium persulfate, present in the suspension medium at a c~ n~ntration of up to about 5 weight percent.
More preferably, the r~ ntr~tion of the initiator is about o . 596 to about 2~ by weight of the aqueous suspension medium.
Production of a ~igh Internal Phase E~nulqion The first step in the production of a HIP~-based microbead is the formation of a high internal phaRe emulsion. A HIPE can be prepared by any of the prior 35 art methods, such as, for example, that disclosed in U. S . Patent No . ~, 522, 953 (Barby et al ., issued June _, _ _ . _ _ _ . . _ _ .. _ .. ....

Wo 95l33553 ~ 2 1 ~q ~7:3 ~ PCrlUS95/06879 ~, 11, 1985), which has been incorporated herein by reference. Briefly, a HIPE is formed by combining the oil and aqueous disnnnt-n--nus phases while subjecting the combination to shear agitation. Generally, a mixing or agitation device such as a pin impeller is used .
The extent and duration of shear agitation must be sufficient to form a stable emulsion. As shear agitation is inversely related to cavity size, the agitation can be increased or decreased to obtain a microbead with smaller or larger cavities, respectively. In one embodiment, a XIPE is prepared using a Gifford-Wood Homogenizer-Mixer (Model 1-LV), set at 1400 rpm. At this mixing speed, the HIPE is produced in approximately 5 minutes. In another embodiment, a HIPE is prepared using an air-powered version of the above mixer (Model 1-LAV), with air pressure set at 5-10 psi for approximately 5-10 minutes. The HIPE can be formed in a batchwise or a rnntlnllmlq process, such as that disclosed in U.S.
Patent No. 5,149,720 (DesMarais et al., issued September 22, 1992).
Production of a HIPE ~icrodroplet Sus~ension Once formed, the HIPE is added to the aqueous gllRrPncinn medium. The HIPE must be added to the 8llYppn~i nn medium in an amount and at a rate suitable for forming a suspension of HIPE microdroplets.
As the HI~E is added, the ~ rPn~inn is subjected to sufficient shear agitation to form a stable suspension. To e~sure that the microbeads produced are relatively uniform in size, the mixing device used should provide a relatively uni~orm distribution o~
agitation force throughout the suspension. As shear agitation is inversely related to microdroplet size, the agitation can be increased or decreased to obtain ~ Wo gs/33s53 2 1 9.b 73 1 pcrlus9sl06879 smaller or larger HIP~ microdroplets, respectively. In this manner, one can control the size of the microbead produced upon polymerization.
To produce a stable microdroplet suspension in a 22 liter spherical reactor having baffles or indents, for example, the HIPE is added to the suspension medium dropwise at a flow rate of up to about 500 ml/minute until the suspension comprises up to about 5096 HIPE.
Agitation can range rom about 50 to about 500 rpm when a propeller- or paddle-style impeller with a diameter of approximately 1. 5 to 3 inches is used. In one embodiment, the HIPE is added to the suspension medium in the 22 liter reactor at a flow rate of 20 ml/minute until the suspension comprises about 109~ HIPE.
Agitation of this mixture at about 250 rpm, followed by polymerization, yields microbeads with an average diameter ranging from about 100 to about 160 ~Lm.
Poly~eri za ti o~ of NIPE Mi crodropl e t~
Once a stable suspension of HIPE microdroplets is obtained, the temperature of the aqueous suspension medium is increased above ambient temperature, and polymerization is initiated. Polymerization conditions vary depending upon the composition of the HIPE. For example, the monomer or monomer mixture and the polymerization initiator (8) are particularly important det~rm;n~ntc of polymerization temperature.
Furth~ ~, the conditions must be selected such that a stable suspension can be ~-;nt~-n.od for the length of time n~r~RS~ry for polymerization. The determination of a suitable polymerization temperature f or a given HIPE is within the level of skill in the art. In general, the temperature of the HIPE suspension should not be elevated above 85~C because high temperatures can cause the s~lcpPnRjnn to break. Preferably, when AIB~ is the oil-soluble initiator and potassium .. ... . , , , _, , , , _ _ _ _ _ , Wo 95/33~3 . ~ 2 1 ~ ~ ~ 7 3 1 . ~ I I U~,5,'0~79 ~
persulfate is the water-soluble initiator, styrene -- ~ are polymerized by ~-tn~inin~ the suspension at 60C overnight (approximately 18 hours).
Wa8hing of the Microbead The polymerization step converts a HIPE
microdroplet to a solid microbead. As discus8ed above, this microbead is generally washed to remove any residual, unpolymerized ~ nnF-nt~ of the HIPE or the suspension medium. The microbead can be washed with any liquid that can solubilize the regidual, Inn~nt~
without affecting the stability of the microbead. More than one cycle of washing may be required. Preferably, the microbead is washed five times with water, followed by acetone extraction for roug_ly a day in a Soxhlet extractor. The microbead can then be dried in any conv.ont-~n~l manner. Preferably, the microbead i8 air-dried for two days or is dried under vacuum at 50C
overnight. The resultant microbeads typically have a bulk density of less than about O.10 gm/ml.
Microbead Production usinq an Oll-Phase Initiator ~i crobead r ^n ts In a second Prhori; r t, a polymeric microbead can be produced by a method wherein the polymerization 1n;~;~tnr i8 pregent in the oil phase instead of the a~ueous phase. Suitable initiators for this embodiment include oil-soluble initiators, such as AIBN, benzoyl peroxide, lauroyl peroxide, VAZ0-type initiators (such a8 l~l'-azobis(cy~lnhpy~npr~rhon;trile~ which is old as VAZ0 cataly8t 88 by Aldrich, Milwaukee, WI), and the 1 ike .
In this ~ ; -t, sufficient initiator is added to the oil phase to initiate a free radical reaction.
Appropriate initiator concentrations for a given microbead preparation can readily be determined by one ~wogsl33553 ~ 73 ~ F~ 8,9 skilled in the art. TypIcallyl the initiator iB
present in a concentration of up to about 5 weight percent of total polymerizable monomer (monomer c, ~ ~nt plus cr~)RRl ink;n~ agent) in the oil phase.
5 The concentration of the ir,itiator is preferably about 0 . 5 to about 3 . 0 weight percent of total polymerizable monomer, more preferably, about 2.2 weight percent.
In addition, the second embodiment requires the use of a stabilizer capable of forming a b~undary 10 between the aqueous disc-~r1t;nll0us phase of the HIPE and the aqueous suspension medium where these two aqueous media interface in the microdroplet suspension. This r~n~ is analogous to the situation in soap bubbles where a boundary formed by detergent molecules 15 separates air on the inside of the bubble from outside air. The st~h; 1 ;~r reduces water loss from the HIPE
microdroplets and helps prevent microdroplet coalescence .
In general, the stabi:Lizer should be a film 20 forming ~ ' that is soluble in organic solve~ts and sufficiently hydrophilic to stabilize tke interface between the aqueous disront; nllous phase of the HIPE and the aqueous suspension medium. These characteristics are found in a variety of natural and synthetic 25 polymers that can serve as stabilizers in this pmho~;r-nt, including cellulose derivatives, such as methyl cellulose and ethyl cellulose, and PVA (less than about 709~ hydrolysis) . Other suitable 5t~h; 1; 7~rS
can be determined empirically by those skilled in the 30 art in accordance with the teachings herein.
Preferably, the st~h; l; 7~r comprises ethyl cellulose.
The stabilizer ~nc~ntration must be suf f icient to reduce water loss from the ~IIPE microdroplets and reduce microdroplet coalescence. Optimal 35 c~nc~ntrations vary with HIPE composition and are det~rm; n~d empirically. Suitable stabilizer _, _ _ , . , . . . . . _ .

Wo 95/33ss3 ~ 9 ~ ) 7 3 ~ r ~ J C C /Y ~
concentrations typically range from about 0 . 01~ to about 159~ by weight of the oil phase. Higher stabilizer concentrations can be employed; however, at concentrations above 1596, the stabilizer is dif f icult to wash out of the polymerized microbeads. Preferably, the stabilizer concentration i8 about 0.1~ to about 1~
of the oil phase, more preferably, about 0.2% to about 0.6~.
If the stabilizer is not soluble in the oil phase, the stabilizer i6 typically dissolved in an inert solvent, and the resulting solution is added to the oil phase of the HIPE . In addition to .snl llh; 1; 7; ng the st~hil; 7Pr~ the inert solvent acts as a porogen. The inert solvent can be any solvent that is capable of solubilizing the stabilizer and that is miscible in the oil phase of the HIPE. Examples of inert solvents useful in the second ~mhnrl;--nt include trichloroethane, toluene, chloroform, and other halogenated solvents, and the like.
Sufficient inert solvent is added to the st~h; 1 i 7~ to increase the solubility of the stabilizer to allow mixing of the st~h; l; zer with the oil phase.
The inert solvent cnnc~ntration varies with the stabilizer and oil phase employed, and the determination of a suitable type and rnnr~ntration of inert solvent to facilitate the mixing of a particular stabilizer with a given oil phase is within the level of 8kill in the art. Typically, the inert solvent concentration ranges from about 39~ to about 609~ by weight of the oil phase and, more preferably, from about 109c to about 4096.
Suitable rnnrpntrations of monomer, crosslinking agent, and ~ ; er are essentially as described above, except that the cnnr~ntrations of any of these components can be reduced to ~c~ ' t~ the inclusion of the oil-soluble polymerization initiator, ~wogs/33553 ~ ? ' ~ q731 PCT/US95/06879 stabilizer, and the inert solvent in the oil phase.
Thus, typical c~ncPntration ranges for these ~ mrr~n~ntc in the secoIld embodiment are as follows:
Concentration -=
Com~onent (weiqht ~ of HIPE) Monomer 4 - 9 o Crosslinker 1 - 89 Emulsifier 3 - 50 Preferred l-~n-~Pntrations for each of these components are the same as discussed above for the first embodiment. In addition, a porogen can optionally be included in the oil phase, as described above for the 15 first P~h",~; t. If a porogen is included, the ron~ ntrations of monomer, crf~c--cl ;nk;ns agent, and/or emulsif ier can be reduced .
The aqueous disrnnt; n~ q phase and the aqueous suspension medium of the second embodiment differ from 20 those of the first embodiment in that the aqueous dis~ nt;n~ us phase and aqueous suspension medium of the second pmho~; t do not include a polymerization ; n; t; ~ t or . In a variation of the second embodiment, the aqueous ~; Cc~nt;nllnus phase consists essentially of 25 water. The aqueous suspension medium of this pmhQrl; comprises a 8-lCFPnrl; ng agent that can be any agent or combination of agents that promotes the formation of a stable suspension of ~IPE microdroplets.
Examples of suitable s~lcpPnA;n~ agents are discussed 30 above. Natural gums, such as, for example, acacia gum (commercially available from Aldrich Chemical Co., Milwaukee, WI), are preferred. The suspending agent can be present in any ~n~ntration that promotes the formation of a stable suspension, typically about 1% to 35 about 309~ by weight of the aqueous suspension medium.

W09SM3553 '. ' `~ 2 1 'hO73 1 P~ 79 ~
Preferably, the suspending agent concentration is between about 236 to about 1596.
Microbead Prepara~ion To prepare microbeads according to the second ~mhotl; t, an oil phase is prepared by combining the oil-soluble polymerization initiator, stabilizer, and inert solvent with monomer, crnssl ;nk;ng agent, and emulsifier. A HIPE is formed by combining the oil and aqueous disrnnt; nllnus phases while subj ecting the combination to suf ficient shear agitation to form a stable irmllR;~rn, as described above. Once formed, the HIPE is added to the ariueous suspension medium in an amount and at a rate suitable for forming a suspension of HIPE microdroplets. AB the HIPE is added, the su8pension is sub~ ected to shear agitation as de8cribed above .
After obtaining a stable suspension, polymerization is initiated by raising the temperature.
A8 rYrl~;n~d above, polymerization conditions vary depending upon the composition of the HIPE, and the determ;n~t;nn of suitable conditions for a given HIPE
is within the level of skill in the art. When lauroyl peroxide is the in;t;~tnr~ for example, polymerization is conve~iently carried out for 20 hours at 50C.
Modifications of the Microbead The microbead is useful for a variety of applications, notably, as an absorbent material and also as a solid support in bioterhnrlor,y applications.
A microbead-based ~hsnrh~nt can be used, for example, to transport solvents, to absorb body f luid8, and as an adhesive microcarrier. Biotechnology ;~rpl;r~t;rnR
include chromatographic separations, solid phase synthesis, immobilization of antibodies or enzymes, and microbial and mammalian cell culture. The basic ~W095l33553 ~ t j; ,7 r~s~ CSs7g microbead can be modified in a variety of ways to produce microbeads that are specialized for particular applications .
Functionalization of the Mi~ro~ead for A~sorotion of Aci ds A wide variety of ionic and polar functional groups can be added to the microbead to produce a polymeric microbead that can absorb large quantities of acidic liquids. Such microbeads generally have a greater capacity to absorb aqueous and/or organic acids compared with their capacity for the neutral oil methyl oleate. In particular, the ratio of aqueous and/or organic acid to methyl oleate absorption i8 generally greater than about 1.2. A preferred starting material for producing such a microbead iB a microbead that is crosslinked from about 1~ to about 50% and has a void volume of greater than about 7096 in its solvent swolle3 state .
The fl-nct;onAl;~ed microbead comprises the structural unit:
[']
in which A represents a crosslinked carbon chain, Y is 30 an optional spacer group, and Z is an ionic cr polar functional group. Z is selected from an amino or substituted amino group and an alkyl cationic ammonium group having eight or more carbon atoms (hereinafter '~a higher alkyl n ) or an alkyl cationic quaternary ammonium 35 group of 8 carbons or les~A in the presence of an organic rollntpri on having 8 or more carbon atoms . The functinnAl;7~l microbead can comprise a single type of Wo 9S/33s53 ~ 2 1 9 ~ 7 3 1 F~~ 79 ~
such structural units or a combination of different types .
In a preferred embodiment, Z is selected from ionic or polar functional groups of structures 1-3:

R2 + H + R4 --N --N X- --N X-\R3 l R4 l \R5 2) ~3 in which R2, R3, R~, R5, and R6 can be the same or different and are selected from an alkyl, cycloalkyl, aryl, and hydroxyalkyl. Alternatively, Rl and R3 can form part of a ring system. When Z is a cationic quaternary ammonium group (3), R", R5, R5 are preferably selected such that the number of carbons present in R4+R5+R6 is 10 or more. When Z i8 an amine salt group (2), R4 and R5 are preferably selected such that the number of carbons present in R~+R5 is 8 or more.
The counterion X for the cationic quaternary ;l~m group (3) or the an amine salt group (2) is an organic or inorganic ion . The counterion f or higher alkyl cationic groups is an inorganic species such as chloride, sulfate, or nitrate. Alternatively, the counterion is a long or short chain organic species such as acetate or oleate.
In another ~ t, R" R5 , and R5 are lower alkyl groups such that the number of carbons present in R4+R5+R6 is less than 10 for a cationic Suaternary ammonium group (3) and the number of carbons present in R4+R5 is less than 8 for an amine salt (2) . In this f~mhof~l t, the counterion X~ is preferably an organic group having 8 or more carbons, such as oleate. For a cationic q-l~t~rn~ry ammonium group (3), X~ can also be O~.

~Woss/33s53 ~ ?~ 1~9~731 r~ o7s The amount of solvent absorbed by the f-lnrtinnAl; 7ed microbead increases with number of ionic or polar functional groups present, provided the level of croRsl;nk;n~ does not exceed approximately 15-20~.
Above this level of cro~Rl; nk; nr~, the amount of liquid absorbed becomes much less sensitive to the degree of substitution since lir~uid uptake is then dependent on the mobility of the solvated polymer chains. The level of crosslinking is controlled by altering the relative rnn~r~ontrations of monomer and crosslinker. Preferably, the level of crosslinking is in the range of about 29 to about 10~6. The degree of funct;rn~l;7~tion is g~nf~rAl ly greater than about 3096, preferably greater than about 5096, and most preferably greater than about 70~.
FunctinnAl; 7ed microbeads are produced by the same methods that are used for producing funct;onAl;7ed HIPE - -polymers. Suitable methods are well known and are disclosed, for example, in U.S. Patent No. 4,611,014 (Jomes et al., issued September 9, 1986), which is incorporated by reference herein in its entirety.
Briefly, the funct;nnAl;7~ microbead is generally prepared indirectly by chemical modification of a preformed microbead bearing a reactive group such as bromo or chloromethyl.
A microbead suitable for subsequent chemical modification can be prepared by polymerization of ~ such as chloromethylstyrene or 4-t-BOC-hydroxystyrene. Other suitable 1 - ~ are styre~e, ~-methylstyrene, or other substituted styrene or vinyl aromatic ~ that, after polymerization, can be chloromethylated to produce a reactive microbead inteL ~ te that can be subseguently converted to a functionalized microbead.
Monomers that do not bear reactive groups (including the croRP~ ;nk;nr~ agent) can be incorporated . _ . . . , , _,, _ _ _ _ Wo9sl33553 ~ 9073~ r~ s~
into the microbead at levels up to about 209~ or more.
To produce HIPE-based microbeads, however, such r ~ s must permit the formation of a stable HIPE.
The rnnr~ntration of the reactive monomer should 5 generally be suf f iciently high to ensure that the functionalized microbead generated after chemical modification bears ionic or polar functional groups on a minimum of about~ 3096 of the monomer residues.
Chemical a; f;cation of the reactive microbead 10 ;ntl ali~t~ is carried out by a variety of conv~nt;nn~l methods. Preferred - 112ry methods for producing amine-, amine salt-, and cationic ~-~t~rn~ry ammonium-fllnrt;nn~1;7ed microbeads are described in detail in r ~/ ~ 2 to 4, respectively.
In another ~mho~a~; t, microbeads bearing ionic or polar groups can be prepared directly by: 1~; f; cation and polymerization of an appropriate subst~nt;~11y water-insoluble monomer.
20 Functionalization of the l~icrobead for Absorotion of A6ueous Solutinnq By srl ~rt; n~ dif ferent polar or ionic functional groups, the microbead can be fllnrt;nn~1;7~ to produce a microbead that absorb6 large riuantities of aqueous 25 ~olllt;nncl and that also acts as an ion exchange resin.
The capacity of these microbeads to absorb a 10~6 sodium chloride solution is such that the ratio of 1096 sodium chloride to water absorption is generally greater than about 0.1, preferably greater than about 0.5, and most 3 o pref erably greater than about 0 . 7 .

~Wo ss/33ss3 ~ i ~-0 7 3 1 PCT/US9S/06879 The microbead functionalized for aqueous absorption comprises the structural unit:
5'~
[-r]
10 in which A is a crn~l; nk~d carbon chain, Y is an optional spacer group, and Z is an ionic or polar functional group. Z is selected from an alkyl cationic ammonium group having ten carbon atoms or less, an alkyl amine salt having eight carbon atoms or lesæ, an 15 alkoxylate, a metal or ammonium or substituted ammonium salt of a sulfuric, carboxylic, phosphoric, or sulfonic acid group, provided that where Z is a sulfonic acid, Y
does not have the structure:

~ CH2~m when m = O
25 The functi~n~l i 7~'d microbead can comprise a single type of such structural units or a combination of different types .
In a preferred embodiment, Z is selected from ionic or polar fllnt~ n~l groups of structures 1-2:
R2X~ + H X~
--N--R3 --N~R2 \R4 ~R3 3 5 ~ 2) in which R2, R3, and R~, can be the same or different and are selected from an alkyl, cycloalkyl, aryl, and hydroxyalkyl. Alternatively, Rl and R3 form part of a .... _ _ _ . .. _ . . _ ~095/33553 ~ 2 ~ ~ 07 3 ~ r~ J,,,5:~6~79 ~
ring system. When Z is a cationic quaternary ammonium group (1), Rz, R3, R4 are preferably selected such that the number of carbons present in R~+R3+R4 is less than 10. When Z is an amine salt group (2), Rz and R3 are 5 pref erably selected such that the number of carbons present in R~+R3 i5 less than 8.
The counterion X~ f or the cationic quaternary ;llm group (1) or the amine salt group (2) is an inorganic species such as chloride, sulfate, or 10 nitrate. Alternatively, the counterion can be a carboxylate species having less than 8 carbon atoms, such as acetate or lactate. For a cationic quaternary ammonium group (1), X~ can also be OH-.
In one embodiment, Z is an alkoxylated chain of 15 the type:
--(OCH2CH) p--OCH~B, where p is 1 to 680, and B - CH--OH or COOM or CE~OSO3M
2 5 ~ Rs Rs and where Rs is a hydrogen or an alkyl group and M is a metal, an ammonium, or a substituted ammonium cation.
Especially preferred is Rs= hydrogen, B = CH2OH, and 30 p~20.
Methods for adding the above-described functional groups to HIPE polymers are di6closed in U. S . Patent No. 4,612,334 (Jones et al., September 16, 1986), which i5 in-,:UI~lOLdLed by reference herein in itE entirety.
35 Microbeads flln~t;nnill;7~cl for aqueous absorption are produced by the same methods that are used for producing the corrP~pnn~; n~ functionalized HIPE
polymers. Suitable methods are the same as those ~Wo9sl33ss3 , ~ t ~ ~ r~ 79 described above for producing microbeads functionAl;
for acid absorption for functional groups of the same basic type (e . g., amine salts) .
In particular, suitable ~ - 2 for producing a 5 reactive microbead intermediate that can be func~irn~l;7ed for absorption of aqueous solutions include chloromethylstyrene, n-butyl methacrylate, t-butyl acrylate, 2-ethylhexyl acrylate, or other appropriate acrylate or methacrylate esters.
10 Additionally, monomers such as styrene, ~-methylstyrene, or other substituted styrenes or vinyl aromatic monomers can be polymerized and then =-chloromethylated, sulfonated, nitrated, or otherwise activated to produce a reactive microbead intermediate 15 which can be aubsequently converted to a funct; nn~ ed microbead. Preferred exemplary methods for producing amine salt-, cationic ruaternary ammonium-, alkoxylate, and sulfonate salt-f1~nct;c~n~1;7ed microbeads are described in detail in ~3xamples 5 to 8, respectively.
P~oduction of a StAh7e rArhOll Struct~lre fr~- the ~i crobeaG7 A microbead can be converted to a porous r~rh~n;ff~rous material that retains the original 25 structure of microbead cavities and interconnecting pores. This material is useful, for example, as a sorption or filtration medium and as a solid support in a variety of bioterhnr~ y appl ;rAt;rn~ (described further in the next section). In addition, the 30 carboniferous microbead can be used as an electrode material in batteries and super-capacitors. Battery electrode materials preferably have a large lattice spacing, such as that of the microbead. A large lattice spacing reduces or ~1 ;minAt~ lattice expansion 35 and contraction during battery operation, extending battery cycle lifetimes. Super-capacitors require _ _ _ _ _ ... . . . . _ _ .. ... _ _ _ _ Wo9s~335s3 I ~ 2 ~ q373 1 r~ 3 ~c~79 , . --highly cnnt~llt t;ve electrodes. The microbead i8 ideally suited for this application because the interconnectedness of the microbead renders it highly conduct ive .
To produce a carboniferous microbead, a stable microbead is heated in an inert at- - s~ht-re as disclosed ~or HIPE polymers in U. S . Patent No . 4, 775, 655 (Edwards et al., issued October 4, 1988), which is incorporated herein by reference in its entirety. The ability of the microbead to withstand this heat treatment varies t1t-rt~nt~1 nt3 on the monomer or r t ~~~ used. Some monomers, such as styrene-based monomers, yield microbeads that must be stabilized against de-polymerization during heating.
The modiîication required to stabilize such microbeads can take many forms. Microbead components and process conditions can be selected to achieve a high level of cross-linking or to include chemical entities that reduce or prevent depolymerization under the heating conditions employed. Suitable stabilizing chemical entities include the halogens; ~3ll1 ft n:~tt-t~; and chloromethyl, methoxy, nitro, and cyano groups. For maximum thermal stability, the level of cross-linking is preferably greater than about 20% and the degree of any other chemical ~l;f;t~tinn is at least about 50%.
St~h;l ;z;n~ entities can be introduced into the microbead after its fnr~t;t~n or by selection of appropriately modif ied monomers .
Once st~h; 1; ~ , the microbead is heated in an inert ~tm~spht~re to a temperature of at least about 500C. To reduce the stabilizer content of the final carboniferous structure, the temperature should generally be raised to at least about 1200C. A
pref erred t~srt~r1 ~ ry method f or producing a stable carbon structure from the microbead is described in detail in Example 9.

~Wo95/33553 ~ 9~731 r~ cr~7s Production of a Microbead ~or Use as a Su~trate Many chromatographic and chemical synthesis techniques employ a substrate. In chromatographic separations, , ~ nnPnt~ of a solution are separated 5 based on the ability of such components to interact with chemical groups linked to the substrate surface.
In solid phase synthesis, the substrate serves as a platform to which a growing molecule, such as a polypeptide, is anchored.
Both processes can be carried out in a batchwise or rr~nt; nl]nus manner . In batchwise processes, the substrate is contained in a vessel, and solutions comprising, . c~ntFI to be separated or reactants are sequentially added and removed by filtration and 15 washing. ~lternatively, in crJntin-~r,us or semicont;n-~rus processes, the substrate is rr,ntqinPd in a column and solutions are seql~PntiAlly passed through the column.
The microbead is uniquely suited for use in such 2 0 systems because the microbead provides a rigid fL . ~L}. that ensures open rh~nnPl ~ for liquid flow and permits solution, -- q or r~artAnt~ to diffuse in and out of the microbead. If desired for a particular application, the microbead can be 25 funct;-~nqli7ed by providing chemical groups at the microbead surface that interact directly with ~ Jllellts or reactants in a solution. Alternatively, the chemical groups at the microbead surf ace can serve as anchors for other reactive species, such as 30 catalysts, enzymes, or Ant;hr.~;P~.
The following sections describe the use of the microbead in chromatography and solid phase synthesis and discuss, as ~ lAry, the functirn~l;7qtion of the microbead for use in ion-exchange chromatography and 35 peptide synthesis. The discussions of these two applications are ;ntPn~ l to be illustrative and do not .... .. . , , ~

w09SI33~3 l ~ 2i 9~731 ~ e~s ~
limit the inventisrl in any way. Variations of these applications will be readily apparent to one skilled in the art and are included within the scope of the invention .

Functionalization of the Microbead for Use in Chromatograsnhy The microbead is useful as a substrate in a variety of chromatographic techniques, lnrlll~;nrJ
10 ion-exchange, gel filtration, adsorption, and aEfinity chromatography .
In ion-exchange c~l r ~ t~-~r,raphy~ the components of a mixture are separated in the basis of dif ferences in net charge. Substrates that separate cations, termed 15 "cation-exchange re8ins'~, are characterized by the presence of negatively charged groups. Conversely, 3ubstrates that aeparate anions, termed "anion-exchange resins", are characterized by the presence of positively charged groups. A ~ -nt that binds to 20 an anion-exchange resin, for instance, is typically released from the resin by increasing the Ph of the column buf fer or ~dding anions that compete with the cs~r-,nPnt for binding to the column. Such a I A-t elutes from the column ahead of, , ^nt- having a 25 higher net negative charge and behind ~ s having a lower net negative charge.
Cation-c~ y~ resins can be produced from microbeads by providing acidic groups on the microbead sur~aces. Suitable groups include the strongly acidic 30 sulfonate group as well as the weakly acidic carboxylate, carboxymethyl, rhnsph-te, sulfomethyl, sul~oethyl, and sulfopropyl groups. Anion-exchange resins can be produced from microbeads by fllnct; rnr l i z; nr, the microbeads with basic groups 3S ranging from the strongly basic qn~t-rn;;ry ~ T"C to weakly basic groups such as the aminoethyl, ~Wo 9sJ33553 .` ; i t ~ 7 ~ ~ 7 3 1 r~ ,,J, e79 diethyaminoethyl, guanidoethyl, and epichlorhydrin-triethanolamine groups.
Acidic or basic groups can be added to a preformed ---microbead as described above for functionalization of 5 the microbead for absorption of acidic and aqueous solutions or by any other convPnt;f~n~l method.
Alternatively, a microbead bearing such groups can be prepared directly by polymerization of an d,~J~J r I~I~J ' iate monomer .
Regardless of the preparation method, the microbead functirn~; 7ed for chromatography generally has a void volume of at least about 709s and a cavity size of up to about 50 ,~m. Preferably microbead has a void volume of about 70~ to about 80g~. This combination of features ensures rapid uptake of fluids, with relatively unobstructed flow through the microbead .
The capacity of the microbead substrate can be increased by adding a gel to the microbead according to the methods disclosed in U.S. Patent ~o. 4,965,289 (~hPrr;n~ton, issued October 23, 1990). As discussed further in the next section! the gel is formed in or added to the microbead cavities and is linked to the microbead surface. The gel bears either acidic or basic groups, depending on whether the microbead substrate is to serve as an anion-e~llallyc~ resin or a cation-exchange resin, respectively.
Functionalization of the Microbead for U~e i~ sOlid Pha~e Synthesis The microbead is fl~nrt~nAl;7~ for use in solid phase synthesis by providing chemical groups linked to the microbead surfacea. The chemical groups are selected to interact with one of the reactants involved in the synthesis. Based on the chemical group selected, microbead substrates can be spP~; ~l; 7ed for _ _ _ _ _ _ .. . . , .. _ . _ . .. . . _ _ _ _ .

Woss/33ss3 ~ . PCrlU595/06 79 q'313~ 8 chemical syntheses of species as diverse as peptides, olignucleotides, and oligosaccharides. Methods for adding appropriate chemical groups to f-lnrt;r,nAli7e the microbead for use in such syntheses do not differ from 5 methods previously described for prior art HIPEs and other polymers.
A microbead can be functionalized for peptide synthebis, for instance, as disclosed for ~IIPE polymers in U.S. Patent No. 4,965,289 (Sherrington, issued October 23, 1990), which is incorporated herein by reference in its entirety. Briefly, a microbead is prepared such that the void volume is at least about 709~ and the cavity size i9 Up to about 50 llm. The microbead is preferably sufficiently cross-linked so that the microbead does not swell to more than twice its dry volume during use.
The chemical group linked to the microbead surf ace can be any group that binds to the :Eirst reactant in the synthesis. In peptide synthesis, for instance, the chemical group is any group that binds the first amino acid of the peptide to be produced. The chemical group should be selected such that the chemical group binds at a position on the amino acid other than the amine group . Typically, the rhPm; rAl group comprises an amine that reacts with the carboxyl group of the first amino acid.
rh~m; rAl groups can be added to a preformed microbead or, alternatively, a microbead bearing chemical groups can be prepared directly by emulsification and polymerization of an appropriate substAnt;~lly water-in~oll-hle monomer. If desired, rh~m;r~l groupg can be further - ~;f;l~l to provide spacer groups that interact with the f irst amino acid of the peptide.
Peptide synthesis is initiated by binding the f irst amino acid to suitable chemical groups on the _ _ _ _ _ ~WO ss/33ss3 t ' ~ 2 ~ 9 0 7 3 ~ PCT/US95/06879 microbead surface. Thé amine groups of the amino acid reactants are generally protected, and thus chain elongation occurs by alternating rounds of deprotection and ''Cllpl; n~ of amino acids . The process is terminated 5 by ~1Pt~ t of the peptide from the substrate, after which the peptide is typically purified.
The capacity of the microbead substrate can be increased by adding a gel to the microbead according to the methods disclosed in U.S. Patent No. 4,965,289 (Sherrington, issued October 23, 1990). Briefly, a suitable microbead is prepared as described above, and a gel or pre-gel is deposited and retained within the microbead cavities. The gel is generally a highly solvent-swollen, cross-linked gel and can, for example, be a soft deformable polyamide gel. For synthetic applications, the gel is generally adapted to interact with a reactant in the synthesis. The ratio of swollen gel to porous material can range from about 60:40 to about 95:5 (weight:weight~ and is more preferably from about 75:25 to about 95:5. The most preferred ratio is a~out 8 0: 2 0 .
The gel can be deposited and retained in the pre-formed microbead by any of the prior art methods for producing gel-filled ~IPE polymers, such as those disclosed in U.S. Patent No. 4,965,289 (Sherrington, issued October 23, 1990), for example. In one embodiment, the gel is formed from pre-gel materials within the microbead cavities . Most pref erably, the gel is retained or anchored in the microbead cavities 3 0 during gel f ormation . The gel can be retained by chain entanglement and/or interpenetration between the gel and the microbead surfaces. In addition, the gel can be retained by a process that is believed to involve chemical binding between the gel and the microbead surfaces. Combinations of the above me~h~n;, are also possible.

W0 95l33553 -- ` 2 ~ q ~ 7 3 1 PCTlUs95/06879 In one embodiment, the microbead i8 contacted with a cnlllt;nn comprising pre-gel c Inn~ntc and a swelling solvent for the microbead. As the pre-gel components p~ --te the microbead and begin to ~orm a gel, the 5 microbead swells, entrapping portions o~ the forming gel by polymer chain interpenetration between the swollen polymeric material of the microbead and the forming gel. Suitable swelling solvents depend on the nature of the microbead polymer and can be readily lo determined by one skilled in the art. The details of a preferred exemplary method for producing a gel-filled microbead by polymer chain entanglement are set forth in ~xample 10.
In another embodiment, retention by chemical 15 bonding can be achieved by reacting the gel or pre-gel ~. _ A'At R with anchor groupg on the microbead.
Suitable gel anchor groups do not differ from those known in the art, ;nr~ ;ng groups that have double bonds available for interaction with a gel or pre-gel 20 c Antc. Such anchor groups can be introduced into the microbead by modification after microbead formation or by ~ler~;r~n of appropriately I a;fiecl i ~~s.
For example, a gel-filled microbead can be produced by reacting an amino methyl-funct;nnAl;z~
25 microbead with acryloyl chloride to produce an acrylated microbead. lrpon heating in the precence of pre-gel, tC to initiate gel polymerization, the double bonds o~ the acrylate group are believed to interact with the f orming gel, resulting in covalent 30 linkage of the gel to the microbead. When an acrylated microbead is employed, suitable pre-gel - ~
include, for example, acryloyl tyramine acetate and acryloyl sarcosine methyl ester. As discussed above, anchor groups can be introduced into the microbead by 35 - ~ ;rAt;nn after microbead formation or by selection of appropriately modif ied s .

~ WO 95/33553 ~ q a 7 3 ~ r~ c~o~79 Use o~ the ~icrobead for InLmo~ilizing l~olecules The microbead can be used as a substrate for a wide variety of molecules including polypeptides and oligonucleotides. The microbead is particularly useful 5 for ;r~ki1;7;ns antibodies, lectins, enzymes, and haptens. Methods for attaching such molecules to polymeric substrates are well known. (See, e.g., Ti jssen, P. LabDratgry T!~hn; c~ueg in Bio~hpm; ~3t~v :-nll MQlecular Bioloqv: Practice alld Theories of ~n7vme 1 o~s~vs New York: Elsevier (1985), at pages 298-314 . ) Several such methods are briefly described below. The methods are described as exemplary and are not ; nt-~n~l.od to be limiting, as one skilled in the art can readily determine how to use or modify these prior art methods to attach a molecule of interest to the microbead.
A polypeptide can be attached to the microbead via non-covalent adsorption or by covalent bonding. Non-covalent adsorption i8 generally attributed to non-specific hydrophobic interactions and ig ;nfl~r~n~ nt of the net charge of the polypeptide. A polypeptide is attached to the microbead by treating the microbead with a buffer solution rrntA;n;nr the polypeptide.
Suitable buffers include 50 Mm r~rh~n~te, pH 9,6; 10 m.M
Tris-HCL, pH 8.5, c~nt~;n;nr 100 mM sodium chloride;
and phosphate buffered saline. Polypeptide crnrrnt~ation is generally between 1 and 10 ~g/ml. The microbead is typically ;nrllh~t~d in this solution overnight at 4C in a humid chamber. Polypeptide 30 adsorption levels can be increased by partial denaturation of the polypeptide, for example, by exposing the polypeptide to high temperature, low pH
(e . g ., 2 . 5 ), or denaturants (e . g ., urea~ .
A number of dif~erent methods are available for 35 the covalent att~ of polypeptides to the micro~ead. Conveniently, a polypeptide having a free _ _ _ _ . . . _ . . . _ . . , _ _ . _ .

wos5/33553 ~ ."l ~l 9 07 3 I P~ C O79 ~
amino group can be attached to a styrene-based microbead, for example, by treating the microbead with glutaraldehyde at low pH. The polypeptide is then added and the pH of the solution is increased to between about 8 . 0 and about 9 . 5 with 100 mM carbonate .
The increase in pH increases the reactivity oi the glutaraldehyde which binds the polypeptide to the microbead surface. Ethylchloroformate can be substituted for or used in combination with glutaraldehyde in the above procedure.
Alternatively, the microbeads can be funct;r,n:ll;7ed for_covalent att~r' - t of polypeptides.
Such a microbead provides anchor groups that bind to the polypeptide of interest. Suitable anchor groups for attaching polypeptides to polymers are well known and include hydrazide groups, alkylamine groups, and Sanger ' g reagent . Anchor groups can be introduced into the microbead by I ';f;r~t;nn after microbead formation or by selection of appropriately modif ied I ~ .
In ~M;t;t~n, a polypeptide can be ~t~r~ to the microbead via a bridging molecule. This method of at~ ' t is useful for polypeptides that bind poorly to the microbead. Such polypeptides are conjugated to a bridrjing molecule that has a high affinity for plastic, such as, for example, bovine serum albumin (BSA). To attach a polypeptide to a microbead via a sSA bridye, BSA is added to the microbead, and then the polypeptide is conjugated to the ~ BSA using a conv~ont;nn~l crosslinker such a~ a carbodiimide.
3 0 In another embodiment, the microbead serves as a substrate for an- oligonucleotide. Conveniently, the nl i~nl~rleotide can be p~F:orhf~d to a microbead by treating the microbead with a polycationic substance such as methylated BSA, poly-~-lysine, or protamine sulfate. When thQ latter is used, the microbead is typically treated for about 90 minutes with a 1 ~Wo 95/33553 " ~ ;. ' 1 PCTIUS95/06879 aqueous solution of protamine sulfate, followed by several distilled water washes. The microbead is then allowed to dry. An oligonucleotide is adsorbed onto the treated microbead by adding the oligonucleotide to 5 the microbead at a concentration of about 10 ~g/ml in a buffer such as 50 mM Tris-HCl, pH 7.5, cnnt~;n;ng 10 mM
ethylPnP~ nPtetraacetic acid (EDTA) and 10 mM
ethylene glycol-bis (~-amino ethyl ether)N,N,N' ,N' -tetracetic acid (E:GTA). Generally, a treatment time of 10 60 minutes provides suitable results.
Use of the Microbead in ~' ~h D~onRitv Cell Cultllre In addition to the above applications, the microbead is also useful in cell culture. Xigh density 15 cell culture generally requires that cells be fed by n nnt;nllmlc perfusion with growth medium. Suspension cultures satisfy this requirement, however, shear effects limit aeration at high cell cnn~entration. The microbead protects cells from these shear effects and 20 can be used in convpnt;nn~l stirred or airlift bioreactors .
To prepare a microbead for use in culturing eukaryotic or prokaryotic cells, the microbead is generally sterilized by any of the many well-known 25 st~-r;l; 7pt inn methods. Suitable methods include irr~ tinn, ethylene oxide treatment, and, preferably, autoclaving. Sterile microbeads are then placed in a culture vessel with the growth medium suitable for the cells to be cultured. Suitable growth media are well 30 known and do not differ irom prior art growth media for suspension cultures. An inoculum of cells is added and the culture is maintained under conditions suitable for cell attachment to the microbeads. The culture volume is then generally increased, and the culture is 35 ~-;nt~;nPd in the same manner as prior art suspension cultures .

W09sl33ss3 ~ 73~ r_~eJ~ 79 Microbeads can be used in cell culture without '; f; ration, however, the microbeads can also be -,~1; f; P~ to improve cell atta, ` - L, growth, and the production of specif ic proteins . For instance, a 5 variety of bridging molecules can be used to attach cells to the microbeads. Suitable bridging molecules include Ant;ho~l;es, lectins, glutaraldehyde, and poly-~-lysine. In addition, sulfonation of microbeads, aa described above, increases cell attArhmPnt rates.
10 Example 11 illustrates the use sulfonated microbeads in an e 1 ilry ~ 1; An cell culture.
This invention is further illustrated by the following specific but non-limiting ~
Procedures that are constructiYely reduced to practice 15 are described in the present tense, and procedures that have been carried out in the l AhnrAtory are set forth in the past tense.
EXAMP~E 1 20 A. Microbead Production Usinq Aclueous-Oil-Pha6e InitiatQrs F ,l:qry pre~erred m;rrnhPArlq were prepared according to the following protocol. The final rnnrPntration of each component of the HIPE and the 25 a5~ueous suspension medium is shown in Table 1, Study 4.
Table 1 also shows studies in which the following protocol was varied as ;ntl;~t~d. The result6 of these studies are set f orth in Table 2 .
l. Prepare an oil phase by ~ ' ;n;nrJ 11.37 gm styrene-based monomer, 11.25 gm DVB, 8.00 gm span 80, 0.27 gm AIBN; and 15.00 gm ~n~ n~ with stirring at room temperature.
2. Prepare an arlueous ~ rrnt;nlln~l~ phase by adding 0.78 gm potassium persulfate to 94.3 ml of distilled water.

o 95/33553 ~ 3 7 3 ~ PCT/US95/û6879 3. Stir the oil phase at approximately 1400 rpm, and then add the aqueous l;Rc~nt;n~1ous phase to the oil phase at a flow rate of 20 ml/minute. Stir the - ` ;n~rl phaaeg at 1400 rpm for approximately 5-10 5 minutes to form a stable HIPE.
4. Prepare an aqueous suspension medium by ;n;n~ 2 gm potassium persulfate and 3 gm of Kaolinite clay (Georgia Kaolin Co., Inc. ) with 1 liter of distilled water. Stir the mixture at 700 rpm for 10 about 15 minutes, and then adjust the stirring speed to 250 rpm.
5. Add the HIPE to the aqueous suspension medium dropwise at a flow rate of 15 ml/minute in a 22 liter Lurex reactor (Model no. LX6214-1008) until the 15 suspension reaches about 2096 HIPE.

6. To form microbeads, polymerize the s~lRp~nRi~n by raising the temperature to 60C overnight (approximately 18 hours) while stirring at 250 rpm.

7 . Wash the resultant microbeads f ive times with 20 water and then perform acetone extraction in a Soxhlet extractor for about a day. Allow the microbeads to air-dry overnight. The bulk density of the resultant material is 0 . 055 gm/ml of dried microbeads.
25 B. Microbead Production IJs;n~ an oil-Ph~Re In;tiator R- l~ry preferred microbeads were E,~pal~d according to the following alternative protocol. The final cnn~.ontration of each component of the HIPE and the aqueous suspension medium is shown in Table 3, 3 o Study 7 . Table 3 also shows studies in which the following protocol was varied as indicated. The results of these studies are set forth in Table 4.
1. In a 1 liter beaker, prepare an oil phase by dissolving o . 4 gm ethyl cellulose in 3 0 gm 35 trichloroethane, and adding 20 gm styrene-based monomer, 19.5 gm DV3, 22 gm span 80, and 0.9 gm lauroyl , . _ . . _ . _ . . _ . . .

wo gs/33553 - ; ~ 2 1~ ~ ~ 7 3 1 PCTrllSg~/06879 peroxide. Mix the r~ ~ nnPnt~: by stirring at room temperature .
2. Place the beaker under a Gifford-Wood Homogenizer-~ixer (Model l-LV; commercially available from Greerco, Hudson, NH) and stir at 1800 to 4800 rpm while slowly adding 850 gm deionized water to form a HI PE .
3. In a 2 liter~glass cylinder reactor (from Ace Glass, Vineland, NJ), prepare an a~ueous suspension medium by combining 120 gm acacia gum with 800 rm of deionized water. While stirring at lO0 rpm, heat the mixture to 60-70C for apprr~r;r-t~oly 15 minutes, and then cool the mixture to about 4 0 C .
4. Add the HIPE to the ar~ueous suspension medium dropwise at a flow rate of approximately 200 ml/minute.
5. Adjust the mixing speed to between 10 rpm and 300 rpm, depending on the desired bead size.
6. To form microbeads, polymerize the suspension by raising the temperature to 45C for appr~nx;r~t~ly 20 hours under constant stirri~g.
7. Wash the resultant microbeads five times with water and then perf orm an acetone extraction f ollowed by a methanol extraction in a Soxhlet ~xtr;lrtnr for approximately 18 hours each. Allow the microbeads to air-dry overnight.

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Wo ss/33ss3 ~ 2 ~,9 0 7 3 ~ / o., r sl~7s Table 4. Properties of Microbeads from Table 3 Studies No- A~ BuLI~ Microbead Size*
Densi~ (g/ml) Range (~m) 0. 15 100-200 2 0.12 80-200 lo 3 0.14 150400 4 0.13 50-150 0.15 50-150 6 o.os 100-300 0.10 30-150 ~ Microbcad yield for each e~periment was ~. 'y 85-95%.
EXl~MPLE 2 Flln~ti9n~1; zation of Microbeads for ~baor~tion of Acids Usincr Amine Grou~s Diethylamine=iunctionalized microbeada are produced from chloromethylstyrene microbeada prepared aa deacribed in Example 1. The microbeada are air-dried overnight and Soxhlet extracted for 15 houra with 200 ml hexane to remove reaidual unpolymeri2ed Cl _ ~nt~. 5 gm of microbeada are then refluxed with 150 ml aqueoua diethylamine for 20 hours. The reaultant diethylamine-f~lnrt;~n~l;7~d microbeads are 859r aubstituted r~nd have a capacity of 1. 5 mM/gm. 1 gm of this material absorbs 20 ml of 1 N sulfuric acid.

~Woss/33ss3 ~ ~ t q073 1 r ~ 79 Fllnrtionalization of Microbeads for Absorl~tion of Acid3 Usinq ~m;n~ ~lts To produce a dihexylammonium salt, dihexylamine-functionalized microbeads are prepared as described above in Example 3 for diethylamine-functir,nAl; 7~.
microbeads . 1 gm dihexylamine-funct; r~n~l; 7ed microbeads are then added to 100 ml methanolic HCl and stirred for 30 minutes. The counterion of the resultant salt ia chloride. The dihexylammonium chloride-functir~nAl i 7e-l microbeads are collected by filtration, washed with 3 times with 50 ml methanol, and air-dried overnight. The resultant microbeads are 70% sub6tituted.
EX~MPLE 4 FuIlctionalization of Microbeads for Absor~tion of Acid8 Uc;nr.
OuaternarY ~mmrn, um GrouT: s To produce a dimethyldecylammonium salt, chloromethylstyrene microbeads are prepared as described in Example 1. The microbeads are air-dried overnight and Soxhlet extracted with hexane to remove residual unpolymerized -nt~. 1 gm microbeads are then filled under vacuum with a 10-fold molar excess of ~th~nrl; c amine and refluxed for 7 hours . The counterion of the resultant salt is chloride. The dime thyl decyl ammonium chl oride - f unct ional i zed microbeads are collected by filtration, washed twice with 50 ml ethanol and twice with 50 ml mc th~n~ l, and then air-dried overnight. The resultant microbeads are 70% substituted.
-W0 95133553 r ~ q f~ 7 3 ~ r~l"J~ 5//!~1?79 Funct lonAl i 7Ation of Microbeads for Ab80r~tion of Aaueous Solutions ~Jsinq Amine IcAl t8 To produce a dimethylammonium salt, diethylamine-5 f1lnrt;r,nA~ 1 microbeads are prepared as described inExample 3. The microbeads are air-dried overnight and Soxhlet extracted with hexane to remove residual unpolymerized ~ n~nt8 1 gm microbeads are then added to 100 ml methanolic HCl and stirred for 10 30 minutes. The counterion of the resultant salt is chloride. The diethylamine chloride-functionalized microbeads are 85~ substituted.

Fl1nrtionalization of Microb~A~q for Absorl~tion of Aqueous Solutir~nq ~Jsinq Oll~ternarY Ammonium Grou~s To produce a dimethyldecylammonium salt, chloromethylstyrene microbeads prepared as described in E:xample 1. The microbeads are air-dried overnight and Soxhlet extracted with hexane to remove residual unpolymerized components. 1 gm microbeads are then treated with lO0 ml aqueous amine for 30 minutes. The resultant dimethyldecyl ;llm chloride-functirnAl i 7ed microbeads are 859~ substituted.

Functi~r~nA]~zAtion of Microbead8 for Absor~tion of Aqueous Solutiong ~8l nr Alkoxvlate Groups Ethoxylated microbeads are prepared from chloromethylstyrene microbeads prepared as described in Example 1. The microbeads are air-dried overnight and Soxhlet extracted with hexane to remove residual = unpolymerized c ^~ts. l gm microbeads are then treated with 100 ml of the anionic form of a --~8 -~wos5/33ss3 ~; ' ~ ` qtJ73 P~ 79 polyethylene glycol (PEG) cnlltA;n;n~ 8-9 ethylene glycol monomers in excess PEG as solvent. The reactants are heated at 95C for 2 hours. The resultant ethoxylated microbeads are 90~ substituted.

~unctionalization of Microbeads for Absor~tion of Aqueous Solutions Usinq SulfQnate Grou~s Sulfonate-functionalized microbeads were produced from styrene microbeads prepared as described in Example 1. The microbeads were dried under vacuum at 50C for two days. 10 gm of microbeads were then added to a 500 ml flask contA;n;ng a mixture of 200 ml of chloroform and 50 ml of chlorosulfonic acid. The flask is shaken at room temperature for two days. The sulfonate-funct;nn~1;7ed microbeads are collected by filtration and washed se~l~nt;Ally with 250 ml each of chloroform, methylene chloride, acetone, and methanol.
The microbeads are soaked in 300 ml 1096 aqueous sodium hydroxide overnight and then washed with water until the eluate reaches neutral pH. The bulk density of the resultant material is 0 . 067 gm/ml of dried microbeads and the capacity is 2 . 5 mM/gm. 1 gm of this material absorbs 23 . 5 gm of water.
EX~MPLE 9 3~roduction of Stable Carbon struct1lre~ from ChloromethYlstvrene Microb~A-l~
3-chloromethylstyrene microbeads are prepared as described in Example 1 such that the level of crosslinking is between 20-409; and the void volume is 90~. 1 gm microbeads are then placed in an electrically heated tube furnace, and the temperature is increased to 600C in an oxygen-free nitrogen rh,~re. The rate of heatillg is generally , .. , .. . . . . . .. . _ _ . , , . _ , _ , _, _ ,, Wo 95/33ss3 ~ 9 0 7 3 1 ~ 79 maintained below 5 C per minute, and in the range of 180C to 380C, the rate of heating does not exceed 2C
per minute. After the heating process, the microbeads are cooled to ambient temperature in an inert ~ ,h~re to prevent oxidation by air.
~XAMP~E~ 1 0 Production of Gel-Filled Microb~
for Yse as a S~lh~trate for Protein Svnthe~is Microbeads with a void volume of 90%, a bulk density of 0 . 047 gm/ml, an average cavity diameter in the range of 1-50_~m, and which are 1096 cr~ l inkP-l are prepared as described in Example 1. The gel employed is poly (N- ~2 - (4-acetoxyphenyl) ethyl) -acrylamide) . To produce a 801ution of gel precur80rs, 2 . 5 gm of monomer, 0 . 075 gm of the crn~sl; nk; n~ agent ethylene bis (acrylamide), and 0 .1 gm of the initiator AIBN is added to 10 ml of the swelling agent dichloroethane.
The gel precursor solution is then deoxygenated by 20 purging with nitrogen.
0 . 7 gm of microbeads is added to the gel precursor solution and polymerization is initiated by heating the mixture at 60C while rotating the sample on a rotary evaporator modified for reflux. The dichloroethane 25 swells the microbeads, allowing the gel precursors to penetrate the microbead and form a polyamide that becomes interpenetrated with the polymer chains of the microbead. After 1 hour, the gel-filled microbeads (hereinafter "composite") are washed with 50 ml 30 dimethyl ft~r~ (DMF) and 50 ml diethyl ether and then vacuum dried. The yield of composite is 2 . 7 gm.
To produce chemical groups within the composite, 0 . 25 gm of the composite is treated with 50 ml of a 5%
8nl -~ n of hydrazine hydrate in DMF for 5 minutes .
35 This treatment provides free phenolic functionalities 21 9~7;~1 ~
~Wo 95l33553 within the gel matrix that act a3 chemical groups for peptide synthesis.
E~AMPL~ 11 Use of Microbeads in Hiqh DensitY Cell Culture To produce microbeads suitable for mammalian cell culture, sulfonated microbeads are prepared as described in 13xample 8 and are then wetted in a 7096 ethanol solution and autoclaved at 121C for lS
minutes. The microbeads are then washed twice with sterile phosphate-buffered saline and once with complete growth medium. 500 mg of the sterile microbeads are placed in a 50C ml roller bottle that has been siliconized to p,Fevent att~rl - t of the cells to the bottle.
An inoculum of 5 x 10~ baby hamster kidney cells in 50 ml of growth medium (r~nt~;n;nrJ 1096 fetal calf serum) is added to the roller bottle. The inoculum is ;nrllh~t~ with the microbeads for 8 hours at 370C with 2 0 periodic agitation to allow cell attachment to the microbeads. The culture volume is then increased to 100 ml, and the roller bottle is gassed with an air-C02 (95:5) mixture and placed in a roller apparatus.
Growth medium is replaced whenever the glucose ~s co~r~n~atior~ drop~ below 1 gm/liter.

Claims (84)

WHAT IS CLAIMED IS:

1. Porous crosslinked polymeric microbeads having cavities joined by interconnecting pores wherein at least some of the cavities at the interior of each microbead communicate with the surface of the microbead and wherein approximately 10% of the microbeads are substantially spherical or substantially ellipsoidal or a combination thereof.

2. The microbeads of Claim 1 wherein the microbeads have an average diameter in the range of about 5 µm to about 5 mm.

3. The microbeads of Claim 1 wherein the microbeads have a void volume of at least about 70%.

4. A process for producing a porous, crosslinked polymeric microbead comprising (a) combining (i) an oil phase comprising (1) a substantially water-insoluble, monofunctional monomer;
(2) a substantially water-insoluble, polyfunctional crosslinking agent;
and (3) an emulsifier that is suitable for forming a stable water-in-oil emulsion; and (ii) an aqueous discontinuous phase to form an emulsion, wherein the emulsion comprises at least about 70% aqueous discontinuous phase;

(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets; and (c) polymerizing the emulsion droplets.

5. The process of Claim 4 wherein the monomer is a styrene-based monomer.

6. The process of Claim 4 wherein the monomer is present in the oil phase at a concentration in the range of about 4 to about 90 weight percent.

7. The process of Claim 4 wherein the crosslinking agent is selected from the group consisting of a divinylbenzenic compound, a di- or triacrylic compound, a triallyl isocyanurate, and a combination thereof.

8 . The process of Claim 4 wherein the crosslinking agent is present in the oil phase at a concentration in the range of about 1 to about 90 weight percent.

9. The process of Claim 4 wherein the emulsifier is selected from the group consisting of a sorbitan fatty acid ester, a polyglycerol fatty acid ester, a polyoxyethylene fatty acid or ester, or the emulsifier is a combination thereof.

10. The process of Claim 4 wherein the emulsifier is present in the oil phase at a concentration in the range of about 3 to about 50 weight percent.

11. The process of Claim 4 wherein the oil phase additionally comprises an oil-soluble polymerization initiator.

12. The process of Claim 11 wherein the initiator is selected from the group consisting of an azo initiator and a peroxide initiator.

13. The process of Claim 11 wherein the oil-soluble polymerization initiator is present in the oil phase at a concentration of up to about 5 weight percent of total polymerizable monomer.

14. The process of Claim 4 wherein the oil phase additionally comprises a porogen.

15. The process of Claim 14 wherein the porogen is selected from the group consisting of dodecane, toluene, cyclohexanol, n-heptane, isooctane, and petroleum ether.

16. The process of Claim 14 wherein the porogen is present in the oil phase at a concentration in the range of about 10 to about 60 weight percent.

17. The process of Claim 4 wherein the aqueous discontinuous phase comprises a water-soluble polymerization initiator.

18. The process of Claim 17 wherein the water-soluble polymerization initiator comprises a peroxide initiator.

19. The process of Claim 17 wherein the water-soluble polymerization initiator is present in the aqueous discontinuous phase at a concentration of up to about 5 weight percent.

20. The process of Claim 4 wherein the oil-in-water suspension comprises an amount of high internal phase emulsion suitable for generating a stable suspension.

21. The process of Claim 4 wherein the aqueous suspension medium comprises a suspending agent.

22. The process of Claim 21 wherein the suspending agent is selected from the group consisting of a water-soluble polymer and a water-insoluble inorganic solid.

23. The process of Claim 22 wherein the suspending agent comprises a water-insoluble inorganic solid that has been modified to increase the hydrophobicity of the inorganic solid particles.

24. The process of Claim 21 wherein the suspending agent is present in the aqueous suspension medium at a concentration of about 1 to about 30 weight percent.

25. The process of Claim 4 wherein the aqueous suspension medium comprises a water-soluble polymerization initiator.

26. The process of Claim 25 wherein the water-soluble polymerization initiator is a peroxide initiator.

27. The process of Claim 25 wherein the water-soluble polymerization initiator is present in the aqueous suspension medium at a concentration of up to about 5 weight percent.

28. The process of Claim 4 wherein the suspension is formed by adding the high internal phase emulsion to the aqueous suspension medium while providing sufficient shear agitation to generate a stable suspension.

29. A process for producing a porous crosslinked polymeric microbead comprising (a) combining (i) an oil phase comprising (1) styrene;
(2) divinylbenzene;
(3) azoisobisbutyronitrile (4) dodecane;
(5) sorbitan monooleate (ii) an aqueous discontinuous phase comprising potassium persulfate to form an emulsion;
(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets, said suspension medium comprising (i) potassium persulfate;
(ii) modified silica (iii) gelatin; and (c) polymerizing the emulsion droplets.

30. A porous crosslinked polymeric microbead produced by the process comprising (a) combining (i) an oil phase comprising (1) a substantially water-insoluble, monofunctional monomer;
(2) a substantially water-insoluble, polyfunctional crosslinking agent;
and (3) an emulsifier that is suitable for forming a stable water-in-oil emulsion; and (ii) an aqueous discontinuous phase to form an emulsion;
(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets; and (c) polymerizing the emulsion droplets.

31. A porous crosslinked polymeric microbead produced by the process comprising (a) combining (i) an oil phase comprising (1) styrene;
(2) divinylbenzene;
(3) azoisobisbutyronitrile (4) dodecane;
(5) sorbitan monooleate (ii) an aqueous discontinuous phase comprising potassium persulfate;
to form an emulsion;
(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets, said suspension medium comprising (i) potassium persulfate;
(ii) modified silica (iii) gelatin; and (c) polymerizing the emulsion droplets.

32. The microbeads of Claim 1 wherein the microbeads have been functionalized such that the functionalized microbeads absorb aqueous and/or organic acidic liquids, said funtionalized microbeads comprising the structural unit:

in which A is a crosslinked carbon chain;
Y is an optional spacer group; and Z is an ionic or polar functional group.

33. The functionalized microbeads of Claim 32 wherein the ratio of aqueous and/or organic acid absorption by the functionalized microbeads to methyl oleate absorption is greater than about 1.2.

34. The functionalized microbeads of Claim 32 wherein Z is selected from the group consisting of an amino or substituted amino group;
an alkyl cationic quaternary ammonium group having 8 or more carbon atoms; and an alkyl cationic quaternary ammonium group of 8 carbon atoms or less in the presence of a counterion that is an organic group containing 8 or more carbon atoms.

35. The functionalized microbeads of Claim 32 wherein Z comprises an ionic or polar functional group selected from the group consisting of species 1-3:
(1) (2) (3) in which R2, R3, R4, R5, and R6 can be the same or different and are selected from the group consisting of an alkyl, cycloalkyl, aryl, hydroxyalkyl; or R2 and R3 form part of a ring system; and the number of carbons present in R4+R5+R6 is 10 or more for a cationic quaternary ammonium (3);
the number of carbons present in R4+R5 is 8 or more for an amine salt (2); and the counterion X? is an organic or inorganic ion.

36. The functionalized microbeads of Claim 35 wherein the number of carbons present in R4+R5+R6 is less than 10 for a cationic quaternary ammonium (3);
the number of carbons present in R4+R5 is less than 8 for an amine salt (2); and the counterion X? is an organic group having 8 or more carbons.

37. The functionalized microbeads of Claim 32 wherein the level of crosslinking is from about 1 to about 20%.

38. The functionalized microbeads of Claim 32 wherein the level of crosslinking is from about 2 to about 10%.

39. The functionalized microbeads of Claim 32 wherein about 95%.

40. The functionalized microbeads of Claim 32 wherein the degree of functionalization is greater than about 50%.

41. The microbeads of Claim 1 wherein the microbeads have been functionalized such that the functionalized microbeads absorb aqueous liquids, said functionalized microbeads comprising the structural unit:
in which A is a crosslinked carbon chain;
Y is an optional spacer group; and Z is an ionic or polar functional group.

42. The functionalized microbeads of Claim 41 wherein the ratio of 10% sodium chloride absorption by the functionalized microbeads to water absorption is greater than about 0.1.

43. The functionalized microbeads of Claim 42 wherein the ratio of 10% sodium chloride absorption to water absorption is greater than about 0.5.

44. The functionalized microbeads of Claim 43 wherein the ratio of 10% sodium chloride absorption to water absorption is greater than about 0.7.

45. The functionalized microbeads of Claim 41 wherein Z is selected from the group consisting of an alkyl cationic quaternary ammonium group having 10 carbon atoms or less;

an alkyl amine salt group having 8 carbon atoms or less;
an alkoxylate group;
a metal, ammonium, or substituted ammonium salt of a sulfuric, carboxylic, phosphoric or sulfonic acid group, except that where Z is a sulfonic acid, Y does not have the structure:

46. The functionalized microbeads of Claim 41 wherein Z comprises an ionic or polar functional group selected from the group consisting of structures 1-2:
(1) (2) in which R2, R3, and R4 can be the same or different and are selected from the group consisting of an alkyl, cycloalkyl, aryl, and hydroxyalkyl; or R2 and R3 form part of a ring system; and the number of carbons present in R2+R3+R4 is less than 10 for a cationic quaternary ammonium (1);
the number of carbons present in R2+R3 is less than 8 for an amine salt (2); and the counterion X? is an inorganic ion or a carboxylate group having less than 8 carbons.

47. The functionalized microbeads of Claim 41 wherein Z is an alkoxylate group having the structure:
where p is 1 to 680, and B = and where R5 is a hydrogen or an alkyl group, and M
is a metal, an ammonium, or a substituted ammonium cation.

48. The functionalized microbeads of Claim 41 wherein the level of crosslinking is from about 1 to about 20%.

49. The functionalized microbeads of Claim 41 wherein the level of crosslinking is from about 2 to about 10%.

50. The functionalized microbeads of Claim 41 wherein the void volume of the microbeads is greater than about 95%.

51. The functionalized microbeads of Claim 41 wherein the degree of functionalization is greater than about 50%.

52. The microbeads of Claim 1 wherein the microbeads have a carboniferous structure.

53. The carboniferous microbeads of Claim 52 wherein the microbeads have a void volume of at least about 90% and a dry density of less than about 0.25 gm/cc.

54. A process for producing carboniferous microbeads comprising (a) producing stable, porous, crosslinked polymeric microbeads having cavities joined by interconnecting pores; and (b) heating said stable microbeads in an inert atmosphere to a temperature of at least about 500°C.

55. A separation method comprising (i) passing a solution having at least two components through porous, crosslinked polymeric microbeads having cavities joined by interconnecting pores, wherein a chemical group that preferentially interacts with one of the two components is linked to the surface of the microbeads; and (ii) separating the two components from one another.

56. The method of Claim 55 wherein the separation method is ion-exchange chromatography.

57. The method of Claim 55 wherein the chemical group is bound to the surface of the microbeads.

58. The method of Claim 55 wherein the chemical group is bound to a gel or pre-gel within the cavities of the microbeads.

59. A method comprising passing a solution including a reactant through porous, crosslinked polymeric microbeads having cavities joined by interconnecting pores, wherein a chemical group that interacts with the reactant is linked to the surface of the microbeads.

60. The method of Claim 59 wherein the chemical group is bound to the surface of the microbeads.

61. The method of Claim 60 wherein the chemical group is bound to a gel or pre-gel within the cavities of the microbeads.

62. The microbeads of Claim 1 additionally comprising a gel or pre-gel within the cavities of the microbeads, wherein the gel or pre-gel comprises a chemical group that interacts with a chemical entity selected from the group consisting of a component of a solution and a reactant in a chemical synthesis.

63. The microbeads of Claim 62 wherein the cavity size is in the range of about 1 to about 100 µm in diameter.

64. The microbeads of Claim 62 wherein the microbeads are crosslinked such that, in the presence of liquid, microbead swelling does not exceed approximately two times the microbead dry volume.

65. The microbeads of Claim 62 wherein the ratio by weight of swollen gel to microbeads lies in the range of about 60:40 to about 95:5.

66. A process for producing microbeads having a gel or pre-gel comprising (a) producing porous, crosslinked polymeric microbeads having cavities joined by interconnecting pores and (b) depositing and retaining a gel or pre-gel in the microbead cavities.

67. The process of Claim 66 wherein step (b) comprises contacting the microbeads with a solution comprising pre-gel components and a swelling solvent for the microbeads for a time sufficient to allow the pre-gel components to permeate the swollen microbeads and form a gel within the microbead cavities.

68. The microbeads of Claim 1 wherein a molecule selected from the group consisting of a polypeptide and an oligonucleotide is attached to the surf ace of the microbeads.

69. The microbeads of Claim 68 wherein the molecule is a polypeptide selected from the group consisting of an antibody, a lectin, an enzyme, and a hapten.

70. The microbeads of Claim 68 wherein the molecule is attached to the surface of the microbeads by a type of attachment selected from the group consisting of non-covalent adsorption, covalent bonding, and attachment via a bridging molecule.

71. A method comprising the steps of, in the following order, (a) contacting cells with porous, crosslinked polymeric microbeads having cavities joined by interconnecting pores in the presence of a culture medium and (b) agitating the microbeads.

72. The process of Claim 4 wherein the oil phase additionally comprises an oil-soluble polymerization initiator, and no polymerization initiator is present in either the aqueous discontinuous phase or the aqueous suspension medium.

73. The process of Claim 72 wherein the oil-soluble initiator is selected from the group consisting of benzoyl peroxide, lauroyl peroxide, and a VAZO
initiator.

74. The process of Claim 72 wherein the oil-soluble polymerization initiator is present in the oil phase at a concentration of up to about 5 weight percent of total polymerizable monomer.

75. The process of Claim 72 wherein the aqueous discontinuous phase consists essentially of water.

76. The process of Claim 72 wherein the emulsion additionally comprises:
(a) a natural or synthetic polymeric stabilizer;
and (b) an inert solvent.

77. The process of Claim 76 wherein the polymeric stabilizer is a cellulose derivative.

78. The process of Claim 76 wherein the polymeric stabilizer is selected from the group consisting of methyl cellulose, ethyl cellulose, and poly(vinyl alcohol).

79. The process of Claim 76 wherein the polymeric stabilizer is present in the oil phase at a concentration in the range of about 0.01 to about 15 weight percent.

80. The process of Claim 76 wherein the inert solvent is selected from the group consisting of trichloroethane and toluene.

81. The process of Claim 72 wherein the suspending agent is a natural gum.

82. A process for producing a porous crosslinked polymeric microbead comprising (a) combining (i) an oil phase comprising (1) styrene;
(2) divinylbenzene;
(3) lauroyl peroxide;
(4) trichloroethane;
(5) sorbitan monooleate (ii) an aqueous discontinuous phase that does not contain a polymerization initiator to form an emulsion;
(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets, wherein said suspension medium comprises acacia gum and does not contain a polymerization initiator;
and (c) polymerizing the emulsion droplets.

83. A porous crosslinked polymeric microbead produced by the process comprising (a) combining (i) an oil phase comprising (1) styrene;
(2) divinylbenzene;

(3) lauroyl peroxide;
(4) trichloroethane;
(5) sorbitan monooleate (ii) an aqueous discontinuous phase that does not contain a polymerization initiator to form an emulsion;
(b) adding the emulsion to an aqueous suspension medium to form an oil-in-water suspension of dispersed emulsion droplets, wherein said suspension medium comprises acacia gum and does not contain a polymerization initiator;
and (c) polymerizing the emulsion droplets.

84. The process of Claim 72 wherein the oil phase additionally comprises a porogen.