Weighted collagen microsponge for immobilizing bioactive materials

Claim 3

The method of claim 1 wherein said thermal soaking is at -5° C. to -60° C. for 2 to 48 hours.

Claim 4

The method of claim 2 wherein said thermal soaking is at -10° C. to -20° C. for 3 to 24 hours.

Claim 5

The method of claim 3 wherein said thermal soaking is at -14° C. for 4 hours.

Claim 10

The method of claim 7 wherein said admixture has a pH less than 3.0.

Claim 13

The method of claim 10 wherein said collagen is Type I.

Claim 14

The method of claim 11 wherein said Type I collagen is selected from the group consisting of tendon collagen and corium collagen.

Claim 16

The method of claim 13 wherein said weighting material has a specific gravity of above about 4.0 and said microsponge has a specific gravity of above about 1.3.

Claim 17

The method of claim 14 wherein said inert weighting material is dispersed throughout said collagen matrix as finely divided powder.

Claim 18

The method of claim 14 wherein said weighting material is centrally disposed as a solid core about which said collagen matrix is formed.

Claim 19

The method of claim 14 wherein said inert weighting material is selected from the group consisting of chromium, tungsten, cobalt, molybdenum, titanium, nickel and alloys thereof.

Claim 20

The method of claim 17 wherein said alloy is 316 stainless steel.

Claim 21

The method of claim 17 wherein said weighting material is an alloy and said alloy is 316 stainless steel and said microsponge is contacted with hybridoma cells to immobilized the cells therein.

Claim 24

The method of claim 21 wherein said vacuum drying is at a pressure less than 3 Torr.

Claim 25

The method of claim 22 wherein said vacuum drying is at a pressure less than 0.2 Torr.

Claim 26

A weighted collagen microsponge produced by the methods in any of claims 1 or 5.

Claim 27

A bioreactor system comprising a plurality of weighted collagen microsponges, wherein said microsponges have been sterilized and aseptically sealed in a reactor vessel and wherein each of said microsponges comprises a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 micron 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 above about 1.05, wherein said weighted collagen microsponges are prepared by a method selected from the group consisting of method 1 and method 2, wherein method 1 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to produce collagen fibers, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) thermally soaking said solid particles by holding said particles at a temperature at which said particles remain frozen and for a period of time and under conditions sufficient to allow partial agglomeration of said collagen fibers to occur, (g) vacuum drying said solid particles into a dry, porous matrix, and (h) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge, and wherein method 2 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to obtain collagen fibers with a diameter less than 1.0 micron, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) vacuum drying said solid particles into a dry, porous matrix, and (g) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge.

Claim 28

A bioreactor system comprising a plurality of weighted collagen microsponges, wherein said microsponges have been sterilized and aseptically sealed in a reactor vessel and wherein each of said microsponges comprises a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 micron 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 above about 1.05, wherein said weighted collagen microsponges are prepared by a method selected from the group consisting of method 1 and method 2, wherein method 1 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to produce collagen fibers, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) thermally soaking said solid particles by holding said particles at a temperature at which said particles remain frozen and for a period of time and under conditions sufficient to allow partial agglomeration of said collagen fibers to occur, (g) vacuum drying said solid particles into a dry, porous matrix, and (h) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge, and wherein method 2 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to obtain collagen fibers with a diameter less than 1.0 micron, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) vacuum drying said solid particles into a dry, porous matrix, and (g) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge.

Claim 29

The biorector system of claim 25 wherein said reactor vessel has a volume between about 0.1 to 10 liters.

Claim 30

The bioreactor system of claim 26 wherein said reactor vessel is a fluidized bed reactor vessel having a fluid distribution plate.

Claim 31

A process for performing a bioreaction comprising (a) immobilizing a bioactive material in weighted collagen microsponges; (b) containing the microsponges having said immobilized bioactive material in a suitable reactor vessel; (c) passing a liquid reagent stream into said reactor in direct contact with said microsponges; (d) agitating the mixture of said microsponges and said reagent stream; and (e) recovering biochemical reaction products from said reactor, wherein each of said microsponges comprises a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 micron to about 150 microns, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said micrsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity above about 1.05, wherein said weighted collagen microsponges are prepared by a method selected from the group consisting of method 1 and method 2, wherein method 1 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to produce collagen fibers, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) thermally soaking said solid particles by holding said particles at a temperature at which said particles remain frozen and for a period of time and under conditions sufficient to allow partial agglomeration of said collagen fibers to occur, (g) vacuum drying said particles into a dry, porous matrix, and (h) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge, and wherein method 2 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to obtain collagen fibers with a diameter less than 1.0 micron, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) vacuum drying said solid particles into a dry porous matrix, and (g) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge.

Claim 32

A process for performing a bioreaction comprising (a) immobilizing a bioactive material in weighted collagen microsponges; (b) containing the microsponges having said immobilized bioactive material in a suitable reactor vessel; (c) passing a liquid reagent stream into said reactor in direct contact with said microsponges; (d) agitating the mixture of said microsponges and said reagent stream; and (e) recovering biochemical reaction products from said reactor, wherein each of said microsponges comprises a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 micron to about 150 microns, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said micrsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity above about 1.05, wherein said weighted collagen microsponges are prepared by a method selected from the group consisting of method 1 and method 2, wherein method 1 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to produce collagen fibers, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) thermally soaking said solid particles by holding said particles at a temperature at which said particles remain frozen and for a period of time and under conditions sufficient to allow partial agglomeration of said collagen fibers to occur, (g) vacuum drying said particles into a dry, porous matrix, and (h) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge, and wherein method 2 comprises the steps of (a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen to obtain collagen fibers with a diameter less than 1.0 micron, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) producing droplets of the weighted acid/collagen mixture of step (c), (e) rapidly solidifying said droplets by freezing to form solid particles of a frozen collagen fiber matrix, (f) vacuum drying said solid particles into a dry porous matrix, and (g) crosslinking the collagen in said dry, porous matrix to produce said weighted collagen microsponge.

Claim 33

The process of claim 28 wherein organisms are the bioactive material immobilized in said microsponges, the microsponges are incubated in said step (d) of agitating the mixture of said microsponges and said reagent stream to promote growth and colonization.

Claim 34

The process of claim 28 wherein organisms are the bioactive material immobilized in said microsponges and the recovered product comprises free organisms which have escaped from said microsponges.

Claim 35

The process of claim 29 wherein said organisms comprise hybridomas and said product comprises monoclonal antibodies.

Claim 36

The process of claim 31 wherein said reactor vessel comprises a fluidized bed reactor vessel.

Claim 37

The process of claim 29 wherein said organisms comprise mammalian cells and said products comprise mammalian cell products.

Claim 38

The process of claim 29 wherein said organisms are genetically engineered microbial cells and said product comprises secreted protein products.

Claim 39

The process of claim 30 wherein said organisms are genetically engineered microbial cells and said product comprises said organisms containing a non-secreted protein product.