CA1275955C - Weighted microsponge - Google Patents

Abstract

WEIGHTED MICROSPONGE Weighted microsponges are described suitable for use in culturing organisms in motive reactor systems. The microsponges have an open to the surface pore structure, and pore sizes and volumes suitable for immobilizing a variety of bioactive msterials. The microsponges also have an average particle size in the range of about 100 to about 1000 microns and a specific gravity above about 1.05.

Description

9~s WEIGHTED MICROSPONGE
This invention was made in the course of, or under, a contract wîth NIH. The governmerlt has rights to the invention pursuant to Grant No. CA37430.

The present invention pertains to the art of immobilizing bioactive materials and particulQrly rel~tes to an improved microsponge for use in motive bioreactor systems. The present invention also relates to the art of culturing microorganisms and cells, hereinafter referred to collectively as organisms, ~nd particularly relhtes to the culturin~ of organisms immobilized on and/or in microsponges in motiYe reactor systems ~s submerged auspensions.
D~ b~ r~
Various arr~ngements for immobilizing bioactive materials are known. ;Solid supports have long been used for immobilizing micro-organisms in the treatment of waste water and related fermentation processes. More recently, solid microcarriers have been used to obtain high cell densities in the culture of attachment-dependent cells. For example, microporous polymeric supports fabricated for example from dextran have been used fo r cultivating cells. Such supports can be obtsinsd commercially from PharmaciQ Fine Chemicals under the brand name Cytodex~. 5uch solid bio-supports? however, are not suitable for motive reaetor systems such as vigorously stirred tanks ~nd fluidized beds slnce substantially all of the cells are adherent to the surface of said supports and thus are exposed to impact stress and trauma during operation.

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Porous inorganic microcarriers also are known and such supports potentially provide protection for the cells in motive applic~tions since the cells populate the interior of the micr~carriers. Unfortunately, inorganic microcarriers cannot be made with th~e proper combination of permeflbility and specific gr~vity to function well in ~ll motive appli-cations. For example, the porous fritted glass or cordierite supports described in Messing et al. U.SO 4,153,510 would typicAlly exhibit spe-cific gravities in ~queous suspension of less than about 1.3 if their void fractions are g~reater th~n about 80% (Note that void fractions for the Messing supports are not disclosedj. Quite understandably, these supports ~re not suitable for all motive reactor systems where a higher speci~ic gravlty generally is needed to ensure high relative velocities for maximum rates of mass transferO Consequently, these supports have generally besn relegated for use in packed bPd applications.
An object of the present invention is to provide a microsponge containing immobilized bioactive materials suitable for use in motive reactor systems.
Another object of the present invention is to provide a micro-sponge suitable for immobilizing a large v~riety of orgRnisms charRcter-ized by wide variations in size and their degree of attachment to solid supports.
A further object of the present invention is to provide a micro-sponge suitable for motive reactor systems which psrmits the continued growth and reproduction of im mobilized organisms It also is an object of the present invention to provide a micro-sponge suitable for motive re~ctor systems which is conducive to maximizing the metabolic activity of immobilized organisms.
Yet another object OI the pressnt invention is to provide a method for continuously culturing organisms at high concentratlons.
Still another object of the present invention is to provide a microsponge suitsble for motive reactor systems which permits the culturing of organisms At high concentrations while accommodating either maximum growth rate or maximum metabolic activity.

~7~9S5 These and other objects of this invention will become apparent from a consideration of the specification and appended claims.
SUMMARY OF THE INVENTION
The present invention pertains to a weighted microsponge for immobilizing bioactive materials in motive bioreactor systems, said microsponge comprising a porous, biostable matrix of a biocompatible polymer containing an inert weighting material, said matrix haring an open to the surface pore structure with an average pore size in the r~nge of from about 1 micron to about 150 microns, with the pores of said rn~trix occupying from ~bout 70 to ~bout 98% by volume of the rnicrosponge, said microsponge also having an Average particle size of from about 100 microns to about 1000 microns and a specific gravity of above about 1.05.
DETAILED DESCRIPTION
___ The present invention is directed to a weighted microsponge pre-pared from a biocompatible polymer containing im mobilized bioactive materisls, particularly organisms~ suit~Me for use in motive bioreactor systems. As used throughout the specification and clsims, the term "bioactive material" broadly encompflsses enzymes and other chemic~l factors such as chelating agents, hormones, antibodies, etc., and organ-isms, i.e., microorganisms and the cells of higher organisms. The organisms may be either living or dead and rn~y be derived without limitation rom such diverse sources 8S bacteria, viruses, fungi, algae, yeasts, animal cells (tissue~, e.g., rnammals, insects and îlsh and plant cells. Since the invention bas particular ~dvantages when used for culturing organisms, it ~enerally will be described with reference to such embodiments, ~lthough it is not to be so-limited.
The microsponge of this inYentiOn is prep~red from a biocompatible (e.g~, non-toxic) polymer th~t is stable in service for an ~ppropriate period of time, e.g.5 on the order of months.
Biocompatibility refers to the ability of the polyrneric (matrix) material to support a Yiable culture of organisms without substantially adversely 5~5~i affecting any desired charQcteristic of the imrnob}lized organisms7 e.g., in the case of hybridomas, the matrix matelrial should not undesirably reduce the production of monoclonal antibodies. The stability or biostRbility of the matrix material refers to its ability to maintain its strength and integrity under in vitro conditions over the relevant time period for culturing the organism of interlsst. ~or example, in the case of a hybridoma culture for producing mono~lonal antibodies, it is expected that the motive bioreflctor would be operated continuously for three to six months or more. Thus, the m~trix material should be biostable for this time period.
Both natural and synthetic polymeric materials can be used as the mfltrix material. Examples of suitable polymers include polysaccharides such as dextran, dextrin, starch, cellulose, agarose, carrageenan and tbe like; proteins such as collagen and the like; flnd synthetic polymers such as polyvinyl alcohsls, polyacrylates, polymeth-acrylates, polyacrylamides9 polyesters, polyurethanes, polyamides and the like~ Generally9 a material's biocompatability and biostability can be verified using routine experimentation.
8ased on its biocompatibility and strength, collagen is presently the material of choice. Collagen is a biodegradable polymer found in animals, including man. It has numerous uses in the medical art and in most applications is reconstituted and crosslinked into an insoluble form using various crosslinking agents, such as aldehydes, e.g., glut-araldehyde and formaldehyde; ethylchloroformate; dimethyl adipimidate;
N,N-methylenebisflcryl~mide; l,Wiscrylamide ethyleneglycol; cyanflmide;
N-N'-diallyltflrtardiamide; cyanogen bromide; concanavalin A; 6-amino-hexanoic acid; 1,6-diaminohexane; succinimidyl active esters;
carbodiimides and compounds having similar crosslinking groups and/or physical treatment techniques such as freeze-drying and severe dehydration at a vacuum of about 50 millitors or more and at a tem-perature ranging from 50C to 200C. Crosslinked collagen has an improved resistance to degradation by collagenAse and other proteinases, ~Z'7~i955 and this is suitable AS ttle biocompatible polymer for the porous matrix of the microsponge.
Crosslinked collagen can be prep~red from both soluble collflgens and insoluble collagens of the Types ~, 11 and 111. The soluble coll~gens are prep~red by limited enzymatic digestion ~nd/or extraction of tissue enriched in such collagen types. Insoluble collagens are derived from the following typical sources: Type I collsgen: bovine, porcine, chicken and fish skin, bovine and chicken tendon hnd bovine and chicken bones including fetal tissues; Type 11 collagen: bovine articul~r cartilage, nasal septum, sternal cartilage; and Type 111 collagen: bovine and human aorta and skin. For example, Type I
collAgen from bovine coriulTI and Type I tendon collagen may be used.
In order to be suitable for culturing high concentrations of organisms in motive reactor systems and allow for the transfer of nutrients to the im mobilized org~nisms and the transfPr of desired products from the microsponge, the microsponge of the present inven-tion must satisfy several functional re~uirements. The microsponge typicfllly is in the shape of a bead and should h&ve ~ particlé size within the range of about 100 microns to 1000 microns, preferflbly from about 200 microns to 500 microns. At larger particle siæes the entire internfll volume of the porous structure is not utilized effectively for producing thè desired product by reaction between the im mobilized bioactive material and the liquid medium contacted therewith, thus de~rading the volumetric productivity of the motive re~ctor employing such microsponges. Smaller particle sizes pre~ent practicfll problems in preparing the microsponge and in operating the motive reactor.
Permeability of the microsponge is another important considerfl-tion. A microsponge's permeability is determined by the inter-relationship of its porosity or void fraction flnd its pore structure.
Yoid fraction is defined as the ratio of the volume of interstices of a material to the total volume occupied by the mflterial and often is 5~5~

expressed ~s a percentage. In order to permit operation at high organism concentrations, the microsponge should have a void fraction OI
between about 70 ~nd 98%. Preferably the void frflction of the microsponge is greater than 85% and most desirably is greater than about 90%.
The microsponge also must possess an open to the surface pore structure. This allows for cell entry, without excessive shear forces, cell retention, subsequent cell growth, and expulsion of exeess cell mas3. For exRmple in cases where the desired product is not secreted by the organisms, e.g., genetically engineered E. coli with a non-expressed rDNA product such as insulin, the organism must be able to escape the microsponge as the immobilized colony expands by division.
An open pore structure is essential if this process is to proceed on a continuous basis, without rupturing the microsponge structure. The desired organism product is recovered as an entrflined component of the culture harvest liquor.
The microsponge should contain pores with Qn average size within the range of about 1 micron for the smalIest microbes and for viruses, up to about 100 microns for large mammalian and plant cells.
Generally, the pores of the microsponge must be at least as large as the smallest m~jor dimension of the immobilized bioactive material but less than about 5 times the largest major dimension. Preferably, the pore size of the mstrix is on the order of 1.5 to 3 times the average diameter of the organism or cell. If unknown, the smallest and largest major dimensions of an organism can be determined using known techniques. Applieants have found that the recited combination of particle sizes and pore sizes insure adequate mass transfer of con-stituents such as nutrients to the immobilized organisms, as well AS
adequate mass transfer of constituents, such as desired metabolites from the immobilized organisms~
For use in motive reactor systems, the microsponge also must be weighted. Polymeric materials suitable for use as the matrix material 12;7S~S~

of the microsponge of the present invention generally have a specific gravity of about 1.0 or less. For proper oper~tion in a motive reactor, ~ specific gravity of above about 1.1)5, preferably above about 1.3 and most preïerably between about 1.~ and 2.0 is desired. It has been found surprisingly thflt it is possible to obtain microsponges of the proper specific gravity using the disclosed biocompatible polymeric m~terials by introducing certain weightirlg additives into the micro-sponge without undesirably reducing its void fraction. The weighting additive must be substantially inert in the reactor environment and non-toxic to the immobilized organisrn, or must be suitably treated to render the additive non-toxic. Also, the weighting additive shculd not adversely affect the productivity of the im mobilized organism . Gen-erally, materials, such as metals and their alloys and oxldes and ceramics, preferably having 8 specific gr~vity above about 4.0 and most preferably above about 7.0 are used. Examples of suitable weighting additives for use in the bro~d practice of the present inven-tion are chromium, tungsten, molybdenurn, cobalt, nickel, titanium and alloys, e.g., Monel, 316 stainless, Vit&lium (~ cob~lt alloy with chro-mium and molybdenum)~ titanium 6Al-4V (~ titanium ~lloy with 6% alu-minum ~nd 4% Yanadium) and H~ynes Stellite Alloy 25 (a cobalt alloy with chromium, nickel, tungsten and rnangenese~. Many of these mate-rials; however, may not be comp~tible with certain organisms and rou-tine experimentation will be necessary to assess toxicity for any appli-cation. For example, in the case of hybridomas titanium is the weighting m~terial of choice, since most other metals are cytotoxic.
The weighting additive can be introduced into and dispersed throughout the microsponge as a finely divided powder, with most particles ha~ing Q si~e on the order of 10 to 40 microns. HoweYer, to minimi2e the surface area o~ the weighting additive, it is desirable to employ it as a solid core In the microsponge. Sufficient weighting material is added to yield a microsponge with the desired specific gr~vityO For examplel about a lO0 micron diameter core of a 75i~5~;

weighting additive h~ving a specific grnvity of RboUt 7.0 coated with a 50 micron thick layer of eollagen having an average pore size of 20 to 40 microns and n void fraction of about 99% yields a microsponge with a specific gravity of about 1.7 hsving an overall void fraction of about 85%. Such a microsponge is particularly suitable for use in an aerobic rnotive reactor system.
Finnlly, for motiYe applications the microsponge should exhibit the proper resistance to nttrition. A charge of microsponges prefer-ably should have a useful llfe on the order OI three to six months or more Typically, the microsponges should e~hibit not greater than about a 10% loss in volume after three months o~ operation.
Normally, organisms exhibit wide variation in their degree of attachment to solid supports. Certain organisms, for examplel readily cling or attach to a wide variety of supports, including both organic and inorganic materials7 while others will only attach to supports of biological origin (attachment-dependent organisms). Other organisms exhibit~ little direct attachment to any support material (attachment-independent organisms). The microsponge of the present invention, because it is prepared from polymeric (organic) materials and because of its permeability (porosity and pore structure) should be suitable for immobilizing substantially ~ll types of organisms~
Any suitable procedure used by the prior art for immobilizing such organisms on microsponges can be used in the present invention including such techniques as adsorption and chemical coupling. For example, in the case of certain organisms it will only be necessary to mix the microsponge in n broth inoculated with the specific organismO
After a short period of time, the organism will coloni~e the micro-sponge and become entrapped In its pores. In the case of some orgnnisms such as fibroblasts and hybridomas, it also may be desirnble to coat the microsponge with attachment-promoting mnterials such ns fibronectin, polylysine and anti-hybridoma antibodies prior to inoculation.
Other techniques can also be used~ such as applying a net chsrge to the surfflce of the microsponge, to enhance im mobili~tion.

s As will be recognized by those skilled in this art, in the broad practice of the present invention9 the procedure used for bringing the im mobilized bioactive material into direet contact wi th a reagent stream such RS a growth supporting medium for culturing of im mobilized organisms is not criti~al and flny of the numerous arrangements available in the prior Qrt including such well known apparatus as stirred tank reactors, fixed bed reactors, fluicli~ed bed reactors and moving bed reactors and the like could be used. Gen-erally, when culturing orgAnisms the microsponges are charged to a suitable reactor and mixed therein with fl nutrient broth and an inoculum o~ the organism. The microsponges should be completely submerged. The microsponges are incubated so that the organisms grow and colonize the porous matrix of the microsponge. Fresh nutri-ent broth fllong with other materials necessary for growth, such as oxygen in the c~se of ~erobic organisms, are supplied in a continuous manner to the reactor and harvest liquor containing the biochemical product of interest is recovered. The biochemical product may eom-prise a primary or secondary metabolite of an immobilized org~nism, excess biom~ss generQted by an immobilized organism containing for e~mple & non-secretory product~ an immobilized enzyme catalyzed reaction product or the like.
A particul~r ~dvantage of the microsponges of the present inven-tion is that tlley can be used in a mixed or motive system such as a nuidized bed reactor. As used herein, the term "motive reflctorl' refers h reactor systems in which relative motion between the micro-sponge ~nd the fluid medium is provided in part by imparting motion to the microsponges themselves. Such reactor systems substantially enhance mass and energy transfer.
To prep~re a microsponge of crosslinked collagen material, a collagen source is formed into a collagen-based solution or dispersion by admixture with A suitable solvent such as fln acid using, for exam-ple, a Waring blender. Next, the weighting ~dditive is blended with .

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the collagen-liquid mixture and the composite mixture is solidi~ied into dry beads using known dry3ng techniques such as sprsy drying, freeze-drying and the like. Any known technique for producing small beads can be employed in carrying out the present invention. Suitable techniques include, inter alia, pressure or air shear spraying, emul-sification techniques, droplet formation using Raleigh liquid jet instability techniques, extrusion techniques, droplet formation using gravity or centrifu~l forces, electrostatic droplet formation, and drop-let formation using inertial forces. For ~example, suitably sized particles have been prepared using inertial forces to for m sm all droplets at the ori~ice of a vibrating needle. Also, larger sized particles possibly could be reduced to the desired p~rticle size by such destructive techniques as grinding and the like. Still additionfll techniques such as rarious coating methodologies, could be used to form microsponges having a solid core of the weighting additive. In this case a shell of the collflgen matrix would surround the weighted core. Those skilled in the art will recogniæe other techniques suitable ~or formin~ sm~ll particles of the types descrlbed above and the present invention is ` not intended to be limited to any specific technique. Finally, the collagen is crosslinked using a suitable treatment as noted above.
Preferably, the microsponges are then sterilized using conventional sterilizaffon techniques and are aseptically packaged for delivery to the ultlmate consumer. The microsponges preferably are sterilized using gamma irradiation. Ethylene oxide aIso may be used as an alternative, as may additional sterilization procedures known to those skilled in the art~ as long as the important characteristics of the microsponge are not compromised. Obviously, when sterilizing the microsponges using ethylene oxide the particles must be thoroughly ventilated in order to remove all traces of this sterilizing agent before subsequently using the microsponges for culturing organisims. To use the sterilized microsponges, the user simply places the microsponges into a previously sterilized reactor, ~dds the proper nutrients and inoculum and ini tiates operfltion. In a preferred embodiment, the package actually comprises a disposable reactor vessel having the necessAry connections for Ieeding a nutrienl st~eam, for removing a harvest liquor and for ancillary operntions, as needed, such as heat exchange, oxygenation and process control. For ~ fluidized bed reactor, the vessel also would contain H suitably designed distribution plate. Such a pre-packllged disposable reflctor vessel m&y have a vol-ume ~etween about 0.1 Iiter and 10 liters. In this case, the user of the reactor simply integrates it with the other process equipment con-sisting of pumps, valves, piping heat and gas exchangers and various instrumentation and related probes and begins operation. Providing Q
disposable reactor, pre-packaged with the microsponges sterilized and ready for use, significantly simplifies st~rt-up procedures for culturing organisms, particularly when changing from one culture to another.
The following example is intended to more fully illustrate the invention ~without acting as a limitation on its scope.

This example describes a suitable rnethod for prep~ring weighted microsponges of crosslinked collagenO Weighted microsponges prepared by this procedure can have particle sizes within the range of about 200 to 800 microns, void fractions o about 80~6, pore sizes on the order of about 20 to 40 microns, and specific gravities on the order of abollt 1.1. The microsponges can be used to support the grourth of hybridoma cells.
Partially purified tendon collagen is milled to obtain small fibers, for example using a Wiley Mill avail~ble from VWR Scientific. The collagen is dispersed into an acidic solution using a Waring blender so as to produce a collagen dispersion having about 1.0% ~by weight) collagen. An inert weighting additive, e.g., titflnium, then is fldded to the collagen dispersion as a fine powder. Frozen droplets of the composite mixture can be formed by flowing the mixture through a ~7~ 5 vibrating hollow needle which discharges into a cryogenic ba$h of liquid nitrogen. The frozen droplets are then vacuum dried, for example using a Virtis Freezemobile Lyophilizer Model 6. After lyophilization, the collagen in the dried microsponges cAn be crosslinked by severe dehydration (dehydrothermal treatment) at a tempersture of about 100C under a vacuum of about ~0 mill~torr for about 72 hours using a drying oqen available from VWR Scientific.
About 300 ml-of the microsponges can be contained in a 600 ml reactor vessel. The microsponges c~n be inoculflted with the hybridoma cells and cultured using ~ suitable nutrient medium. The reactor can be operated at a solids concentration of about 25~40%, as the content of the reactor is vigorously agitated. A nutrient medium such as Delbecko Modified Eagle medium with 10% fetal calf serum can be passed into the reactor in a continuous manner and a product stream containing the monoclonal antibodies can be recovered at a substflntially equivalent flow rate.
It will be obvious to one of ordinary skill that numerous modifications may be ~m~de without departing from the true spirit and scope of the invention whi~ch is to be limited only by the appended claims.

Claims (20)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A weighted microsponges for immobilizing bioactive mate-rials in motive bioreactor systems, said microsponge comprising a porous, biostable matrix of a biocompatible polymer containing an inert weighting material, said matrix having an open to the surface pore structure with an average pore size in the range of from about 1 to about 150 microns, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said microsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity of above about 1.05.

2. The microsponge of claim 1 having immobilized therein bioactive material selected from the group consisting of enzymes, microorganisms, dead cells and living cells.

3. The microsponge of claim 1 wherein said biostable, biocompatible polymer is selected from the group consisting of collagen, cellulose, dextran, dextrin, polyamides, polyesters, starch, agarose, carrageenan, polyurethanes, polyvinyl alcohols, polyacrylates, polymethacrylates and polyacrylamides.

4. The microsponge of claim 1 wherein said inert weighting material is selected from the group consisting of metals, metal alloys, metal oxides and ceramics.

5. The microsponge of claim 4 wherein said weighting mate-rial has a specific gravity of above about 4.0 and said microsponge has a specific gravity of above about 1.3.

6. The microsponge of claim 5 wherein said inert weighting material is dispersed throughout said porous matrix as finely divided powder.

7. The microsponge of claim 5 wherein said weighting mate-rial is centrally disposed as a solid core about which said porous matrix is formed.

8. The microsponge of claim 5 wherein said inert weighting material is selected from the group consisting of chromium, tungsten, cobalt, molybdenum, titanium, nickel and alloys.

9. The microsponge of claim 8 wherein said weighting mate-rial is titanium and said microsponge has hybridoma cells immobilized therein.

10. A bioreactor system comprising a plurality of the weighted microsponges of claim 1 sterilized and aseptically sealed in a reactor vessel.

11. The biorector system of claim 10 wherein said reactor has a volume between about 0.1 to 10 liters.

12. The reactor system of claim 11 wherein said reactor is a fluidized bed reactor, having a fluid distribution plate.

13. A process for performing a bioreaction comprising:
immobilizing a bioactive material in the weighted microsponges of claim 1; containing the microsponges having said immobilized bioactive material in a suitable reactor vessel; passing a liquid reagent stream into said reactor in direct contact with said microsponges; agitating the mixture of said microsponges and said reagent stream and recovering the biochemical reaction products from said reactor.

14. The process of claim 13 wherein organisms are immobilized in said microsponges, the microsponges are incubated to promote growth and colonization of said microsponges by said organ-isms, said reagent comprises nutrient media for promoting the growth and metabolism of said organisms, and wherein said product comprises metabolites of said organisms.

15. The process of claim 13 wherein organisms are immobilized on said microsponges and the recovered product comprises free organisms which have escaped from said microsponges.

16. The process of claim 14 wherein said organisms comprise hybridomas and said product comprises monoclonal antibodies.

17. The process of claim 16 wherein said reactor vessel com-prises a fluidized bed reactor.

18. The process of claim 14 wherein said organisms comprise mammallan cells and said products comprise mammalian cell products.

19. The process of claim 14 wherein said organisms are genetically engineered microbial organisms and said product comprises secreted protein products.

20. The process of claim 15 wherein said organisms are genetically engineered microbial cells and said product comprises said cells containing a non-secreted protein product.