WO1996039432A1

WO1996039432A1 - Avian immunomodulator product and method of making the same

Info

Publication number: WO1996039432A1
Application number: PCT/US1996/008351
Authority: WO
Inventors: James E. Hutchins; Jeffrey L. Pfohl; Julius K. Tyczkowski; Richard S. Girardi; Vivian W. Doelling
Original assignee: Embrex, Inc.
Priority date: 1995-06-06
Filing date: 1996-06-03
Publication date: 1996-12-12
Also published as: ZA963309B; AU5967296A

Abstract

An avian immunostimulant product containing Interleukin-2 is produced from an avian spleen cell conditioned medium, with the product essentially free of proteins having a molecular weight greater than 100 kilodaltons, and preferably with the product essentially free of proteins having a molecular weight less than 10 kilodaltons is disclosed. The product is preferably provided in sterile form. Methods of making the immunostimulant product are also disclosed.

Description

AVIAN IM UNOMODULATOR PRODUCT AND METHOD OF MAKING THE SAME

Field of the Invention The present invention involves methods for the large-scale manufacture of a stable immunomodulator product containing avian Interleukin 2 from a spleen cell conditioned medium.

Background of the Invention

Interleukin 2 (IL-2) , previously designated T- cell growth factor, provides a stimulus for the proliferation of activated T lymphocytes. See generally O. Vainio et al. , Scand. J. Immunol . 23, 135-142 (1986) . It is a known component of mitogen-stimulated chicken spleen cell conditioned medium (CM) , See K. Schauenstein et al., Dev. Comp . Immunol . 6, 533-540 (1982) ; see also T. Hovi et al., Cellular Immunol . 39, 70-78 (1978) ; H. Weiler and V. Von Bulow, Vet. Immunol . Immunopathol . 14, 257-267 (1987) ; G. Kromer et al . , J. Immunol . Methods 73, 273-281 (1984) .

U.S. Patent No. 5,034,513 to T. Fredericksen and J. Sharma describes an avian IL-2 obtained from conditioned medium. The purification method employed concentration by vacuum dialysis, hydrophobic interaction chromatography, anion exchange chromatography, and high resolution gel filtration. The method is not easily adapted to the large-scale manufacture of an IL-2 containing product. Other characterizations of avian IL- 2 are provided in M. Schnetzler et al. , Eur. J. Immunol . 13, 560-566 (1983) and T. Myers et al . , Vet . Immunol . Immunopathol . 34, 97-114 (1992) .

U.S. Patent No. 5,106,617 to T. Fredericksen and J. Thaxton describes methods of treating immature birds, including birds in ovo, with IL-2. The IL2- containing product employed was prepared by centrifugation, concentration on lOkDa molecular weight cut-off membranes, HPLC gel filtration of the resulting supernatant with a G2000 SW TSK column, and then concentration with a CENTRICON™ concentrating device having a lOkDa molecular weight cutoff filter. While this provides a more convenient method of preparation than that described in T. Fredericksen and J. Shar a, supra, it is nevertheless desirable to have an economical method for the large-scale manufacture of a purified conditioned medium containing avian IL-2 which provides a uniform and stable product.

Summary of the Invention

A first aspect of the present invention is an avian immunostimulant product produced from avian spleen cell conditioned medium, with the product essentially free of proteins having a molecular weight greater than

100 kilodaltons, and preferably with the product essentially free of proteins having a molecular weight less than 10 kilodaltons. The product is preferably provided in sterile form.

A second aspect of the present invention is a method of making a concentrated, sterile, avian immunostimulant product containing Interleukin 2. The method comprises:

(a) providing a plurality of avian spleens; then

(b) mincing the plurality of avian spleens in an aqueous solution to provide a minced spleen slurry; then

(c) homogenizing the minced spleen slurry to provide a crude dissociated spleen cell suspension; then

(d) separating tissue and red blood cells from the crude dissociated spleen cell suspension to provide a purified dissociated spleen cell suspension; then (e) incubating the purified dissociated spleen cell suspension in a culture medium in a tissue culture vessel, and preferably in the presence of a mitogen, to produce a spleen cell conditioned medium; then (f) removing cells from the conditioned medium to provide an essentially cell free conditioned medium; then

(g) separating proteins having a molecular weight greater than 100 kilodaltons from the essentially cell free conditioned medium to produce a dilute immunostimulant product; then

(h) separating proteins having a molecular less than 10 kilodaltons from the dilute immunostimulant product to produce a concentrated avian immunostimulant product; and then

(i) sterile filtering the concentrated avian immunostimulant product to provide a sterile concentrated avian immunostimulant product.

A third aspect of the present invention is a sterile concentrated avian immunostimulant product produced by the method given above.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.

Detailed Description of the Invention

Spleens from any avian species can be employed in carrying out the present invention, including chicken, turkey, duck, geese, quail, ostrich, and pheasant spleens. Chicken spleens are currently preferred. The spleens are preferably provided in capsulated form to reduce the possiblity of contamination thereof during shipping. The spleens are typically shipped refrigerated

(on ice) in containers containing an aqueous antibiotic solution. Once the appropriate spleens are provided, the spleens are minced in an aqueous solution to provide a minced spleen slurry.

The minced spleen slurry is homogenized, gently, to provide a crude dissociated spleen cell suspension. In a preferred embodiment of the invention, this homogenizing step is a dispersion, or "mixing", homogenization step, preferably carried out on a Seward Medical STOMACHER 400™ laboratory blender. The term "mixing, " as used herein, has its conventional meaning in the art, refering to a treatment characterized by vertical and lateral flow without swirl or surface vortex. See McGraw-Hill Encyclopedia of Science and Technology, Vol. 11, pg 303-305 (7th Ed. 1992) . In the STOMACHER 400™ laboratory blender, the slurry to be dispersed is placed in a closed rectangular bag, the bag mounted in the blender, and the bag alternately compressed and decompressed by a paddle that contacts the outer side portion of the bag. The side surface area of the bag is greater than the surface area of the paddle that contacts the bag. Hence, compression of the bag by the paddle causes the slurry in the bag to flow away from (or be displaced from) the portion of the bag underlying the paddle, and decompression of the bag by the paddle allows slurry in the bag to flow into the portion of the bag underlying the paddle. This is carried out until clumps of cells are eliminated and the slurry becomes a uniform, essentially homogeneous, suspension of cells.

Following homogenization, tissue and red blood cells are then separated from the crude dissociated spleen cell suspension to provide a purified dissociated spleen cell suspension consisting essentially of white blood cells (preferably with less than five percent red blood cells to facilitate subsequent processing thereof) . Tissue and red blood cells can be separated by any suitable means, with differential centrifugation currently preferred. The purified dissociated spleen cell suspension has (or is adjusted to have by dilution with additional culture medium) from .2 to 3 x 10⁷ viable white blood cells/ml (more preferably .5 to 1.5 x 10⁷ viable white blood cells/ml, and most preferably 1 x 10⁷ viable white blood cells/ml) .

The purified dissociated spleen cell suspension is incubated in a suitable culture medium in a tissue culture vessel, which tissue culture vessel is placed in an incubator, to produce a spleen cell conditioned medium. The culture medium optionally, but preferably, contains a mitogen such as concanavalin A (Con A) in an amount of from 1 to 10 μg/ml, and more preferably from 2.5 to 5 μg/ml. The tissue culture vessel preferably includes a wall that is impermeable to liquid, permeable to oxygen and permeable to carbon dioxide, such as a flexible plastic bag. Suitable bags are known in the art and are described in, for example, U.S. Patent No. 4,140,162 to Gajewski et al . and U.S. Patent No. 4,227,527 to DeFrank et al. (the disclosures of all U.S. Patent References cited herein are to be incorporated herein by reference) . A specific suitable bag is the FENWAL^® LIFECELL^® Tissue Culture Flask, Code 4R2113, available from Baxter Healthcare Corp., Fenwal Division, Deerfield, Illinois 60015 USA. , which provides 750 cm² of surface area when stored flat.

While numerous different culture conditions will be readily apparent to those skilled in the art, optimum results are obtained when the culturing step is carried out in an atmosphere of from 6.5 to 8.5 percent carbon dioxide (by volume) (with the balance of the atmosphere being any suitable oxygen-containing composition of gas or gases, such as air, air enriched with oxygen, etc.) . An atmosphere of 7.5 percent carbon dioxide is currently preferred. The atmosphere is preferably saturated with water vapor. Incubation is preferably carried out at a temperature of from 37°C to 43°C, more preferably 41°C to 43°C, and most preferably at 42°C. Incubation may be carried out from one to three or four days, with an incubation period of one and one half days to two and one half days currently preferred. After incubation, cells are removed from the spleen cell conditioned medium to produce an essentially cell-free spleen cell conditioned medium (e.g., removal of 95% or 99% or more of cells) . Removal of cells can be carried out by any suitable means, such as continuous flow centrifugation, tangential flow filtration, non- continuous flow centrifugation, etc. Continuous flow centrifugation is currently preferred.

Optionally, but preferably, the step of removing cells is followed by the step of removing particles of a size greater than .5 microns from the essentially cell-free spleen cell conditioned medium.

Following cell removal (and the optional particle removal step noted above) , the next step involves separating proteins having a molecular weight greater than 100 kilodaltons from the conditioned medium to produce a dilute immunostimulant product; and then separating proteins having a molecular weight less than 10 kilodaltons from the dilute immunostimulant product to produce a concentrated avian immunostimulant product. Concentrated avian immunostimulant products of the present invention are, as noted above, essentially free of proteins having a molecular weight greater than 100 kilodaltons. By "essentially free," it is meant having been processed to essentially exclude proteins over 100 kilodaltons, though the remaining proteins may aggregate over time to form entities having molecular weights greater than 100 kilodaltons. In a preferred embodiment the product is essentially free of proteins having a molecular weight less than 10 kilodaltons (i.e., consists essentially of avian spleen cell conditioned medium proteins having molecular weights between 10 and 100 kilodaltons) . The product is preferably a sterile product, with sterility being achieved by sterile filtration as given above with subsequent sterile conditions in accordance with known procedures. For example, sterile filtration may be carried out with a sterile filter having a pore size of .45 microns, but is preferably carried out with a sterile filter having a pore size of .2 microns, and may optionally even be carried out with a sterile filter having a pore size of .1 microns. The sterile product is preferably packaged in a suitable container, such as a polypropylene bottle or vial. The packaged sterile product is typically an aqueous product, which may be provided in liquid or frozen form. Alternatively, the liquid product may be lyophilized to provide a lyophilized product in accordance with known techniques. The product has a shelf life of at least 3 months under refrigerated conditions (i.e., 4°C) , and more preferably has a shelf life of six months or one year under refrigerated conditions. The product has a shelf life of at least three months at room temperature (i.e., 25°C) .

Products of the present invention are useful for inhibiting early mortality due to Escherichia coli infections in chicks, are useful as a source of natural cytokines such as avian IL-2, and are useful as an intermediate for the manufacture of natural cytokines such as purified avian IL-2.

The present invention is described in greater detail in the following non-limiting Examples.

EXAMPLE 1 Aseptic Removal of Spleens for

Production of Avian Conditioned Medium (CM)

Spleens removed from specific pathogen-free

(SPF) chickens are used to provide immune cells that are stimulated to produce CM. As cells from these spleens are cultured for one and one-half to two and one-half days in tissue culture media, low level contamination in the spleens can result in CM heavily contaminated with microorganisms. Therefore, it is important that the spleens are removed using aseptic techniques and shipped under conditions that would minimize contamination. This example describes spleen excision techniques that minimize contamination in the spleens.

Spleens are excised using aseptic techniques on a clean work bench sanitized with either 70% ethanol, 2% sodium hypochlorite, or other disinfectant. Solution preparation for spleen excision and washing of spleens is performed in a laminar flow hood. Only birds healthy in appearance are used for excision.

To harvest the spleens, up to twelve (12) birds are euthanized at a time by either asphyxiation with C0₂ or cervical dislocation. Birds with rigor mortis are not used. Spleens are not harvested if the bird's digestive system has been ripped or torn. Only normal-looking spleens are harvested from birds. Spleens are harvested promptly and placed into a bottle of phosphate buffered saline plus gentamicin sulfate (50 μg/mL) and streptomycin sulfate (100 μg/mL) (PBS + antibiotics) .

Following harvesting of the spleens, all washing procedures are performed under the laminar flow hood. The spleens are gently swirled in the bottles containing PBS + antibiotics, after which the PBS + antibiotics is poured into a waste container. This washing step is repeated two times. The washed spleens are then poured into a bottle of IX Hanks' Balanced Salt Solution (HBSS) plus antibiotics (same as above) . The remaining headspace in the bottle is filled with IX HBSS plus antibiotics.

After washing, containers of spleens are placed into a refrigerator until ready for transport on ice to the production site. EXAMPLE 2

Preparation of Conditioned Medium

Splenocytes for concanavalin A stimulation are isolated from SPF chicken spleens, which are processed at the manufacturing facility within 30 hours of spleen excision. A single cell suspension of 1 x 10⁷ white blood cells with less than 5% red blood cells (RBC) is obtained by mincing the spleens and homogenizing and centrifuging the minced spleen tissue. The cell suspension is incubated in DMEM Production Medium (Dulbecco's Modified Eagle's Medium with 2.2 g/ l sodium bicarbonate supplemented with 110 mg/ sodium pyruvate, 292 mg/*f L- Glutamine, 50 μq/ t gentamicin sulfate and 100 μg/m£ streptomycin sulfate) in the presence of concanavalin A (Con A) (2.5μg/m-0 at 42°C with 7.5% carbon dioxide in tissue culture bags or other cell culture vessels for one and one-half to two and one-half days (we currently incubate for 42 hours) . The following procedures are performed using aseptic technique under a laminar flow hood, with all reagents at room temperature.

A. Cell Preparation Spleens are received on ice in bottles and maintained on ice until processing. After the spleens are washed in PBS + antibiotics, they are transferred to sterile petri dishes and weighed. Dissociation Medium (Alsever's solution supplemented with 50μg/m gentamicin sulfate and 100 μg/m£ streptomycin sulfate and 1% Lipid Concentrate) is added to the spleens. The spleens are minced without decapsulation into a uniform slurry with a razor blade mincer (a polymeric block with parallel spaced-apart razor blades and a polymeric handle inserted therein) . The minced tissue is suspended in Dissociation Medium. The suspended tissue is poured into a large STOMACHER™ laboratory blender bag. After placing the bag into another sterile bag, the bags are inserted in a Seward Model 400 STOMACHER™ laboratory blender for homogenization of the tissue therein. The homogenized tissue suspension is filtered through a perforated disk into centrifuge tubes. The tissue is centrifuged for 5 minutes at 120 x g. The supernatants (including cells) are poured into clean centrifuge tubes. The tissue pellet from each tube is retained. Each tissue pellet is resuspended in Dissociation Medium. The resuspended tissue is centrifuged for five minutes at 120 x g. The supernatant is combined with previous supernatants and centrifuged for five minutes at 1200 x g.

Each resultant pellet is resuspended in DMEM Production Medium (DMEM) . An equal volume of suspension is placed into 50m tubes, approximately 45m per tube. The tubes are centrifuged for five minutes at 1200 x g. The clear supernatant is removed and discarded. The white cell suspension, above the red cell pellet, is removed and placed in a bottle with DMEM. The remaining red cell pellets are resuspended in DMEM and placed into 15m.? tubes, 10-15 mi per tube. Each tube is centrifuged for five minutes at

1200 x g and the process of removing the white cell suspensions is repeated. The white cell suspension is added to the previously collected white cell suspension and white blood cells (WBC) and red blood cells (RBC) counted.

The red cell pellets are resuspended in DMEM, pooled and counted. The volume of RBC suspension required to obtain up to 5% RBC in the final cell preparation is added to the white cell suspension. B. Cell seeding. DMEM is warmed to 42°C. Con

A is added to the warmed DMEM to obtain a concentration of 2.5 μg/mt . The amount of the final cell preparation required to achieve a cell density of 1.0 x 10⁷ viable WBC/mf , and not greater than 5% red blood cells (RBCs) , is distributed to the tissue culture bags (TCBs) or other cell culture vessels and incubated at 42°C with 7.5% carbon dioxide for approximately one and one-half to two and one-half days (we currently incubate for 42 hours) .

EXAMPLE 3 Removal of Cells from Culture Media using Continuous Flow Centrifugation

After incubation, the conditioned medium is drained into large containers (carboys) . A peristaltic pump is used to transfer conditioned medium through a continuous-flow centrifuge (a Beckman model J2-MI centrifuge with a Beckman JCF-Z continuous flow rotor) , sanitized with 100 ppm hypochlorite solution. Cells are removed, and the cell-free conditioned medium is collected and set aside for the next step of the purification phase (Example 4 below) .

EXAMPLE 4

Microfiltration of Cell-free Conditioned Medium The cell-free conditioned medium is pumped through Gelman ABSOLIFE™ 1.2μm, 0.6μm and SUPORCAP™-100 0.8/0.45μm capsule filters for clarification and prefiltration. The filters are attached serially and cell free conditioned medium is pumped through the filters beginning with the 1.2μm Absolife filter. When the operation is complete, filters may be rinsed with 0.4M NaCl to increase recovery of the desired product.

EXAMPLE 5

Small Scale Ultrafiltration of Conditioned

Medium to Produce Immunomodulator Product After conditioned medium containing IL-2 is microfiltered to remove cellular debris (see Example 4) , the filtered conditioned medium is then processed through the ultrafiltration (UF) system to produce a concentrated

10-100 kilodalton (kDa) cut. Also, during the UF process, low molecular weight media components, including antibiotics, are washed away and the buffer replaced with a specially formulated phosphate buffer.

For small-scale ultrafiltration of the conditioned media, the Amicon CH2 Ultrafiltration system is used according to routine operating procedures. The Amicon CH2 is sanitized by recirculation with 100 ppm hypochlorite for 1 hour followed by successive washing twice with deionized water, once with PBS and once with deionized water. Post operation cleaning of the CH2 consists of three consecutive washes of 0.IN NaOH, followed by 100 ppm hypochlorite (1 hour recirculation) , and 0.05N NaOH storage.

A. Production of ≤lOOkDa filtrate. For the production of ≤lOOkDa filtrate, an S1Y100 (Amicon) ultrafiltration cartridge is used in the CH2 system. The conditioned medium microfiltrate is ultrafiltered according to routine operating procedures. The >100 kDa retentate volume is reduced to minimum. (System volume for S1Y100 is approximately lOOv i . ) 200m of 0.4M NaCl is added to the >100 kDa retentate, and the solution is put through the filtration process again. The ≤IOO kDa filtrate is collected and combined with previous filtrate. This process is repeated again with the remaining >100 kDa retentate. B. Production of 10-100 kDa cut. To obtain the 10-lOOkDa cut, an S1Y10 (Amicon) ultrafiltration cartridge is used in the CH2 system. The ≤lOOkDa permeate is recirculated through the lOkDa ultrafiltration system and the retentate concentrated to 2-10% of the volume of the lOOkDa permeate. The retentate is washed 1-2 times with two to three volumes of Final Buffer (0.1M potassium phosphate, pH 7, 0.1% trehalose and 0.1% glycerol) . After each washing, the retentate is concentrated to its original volume in the lOkDa ultrafiltration system. The 10-lOOkDa concentrate is drained into a sterile tube and the ultrafiltration cartridges and reservoir are rinsed with Final Buffer, and the rinse is combined with the concentrate.

After processing, the immunomodulator product is sterile-filtered as described in Example 7 below.

EXAMPLE 6

Large-Scale Ultrafiltration of Conditioned Medium to Produce Immunomodulator Product

The Amicon SP20 ultrafiltration system is used for large-scale ultrafiltration of the conditioned medium. Molecular weight cut-offs used in this procedure are lOOkDA and lOkDa. The filtration described in this example is "large-scale" in the sense that it can be used to process up to approximately 100-*?. The SP20 is sanitized by recirculation with 100 ppm hypochlorite for 1 hour followed by successive washing twice with deionized water, once with IX PBS and once with deionized water. Post operation cleaning of the SP20 consists of three consecutive washes with 0.IN NaOH, followed by 100 ppm hypochlorite (1 hour recirculation), and 0.05N NaOH storage.

A. lOOkDa ultrafiltration. production of < lOOkDa filtrate. For the production of ≤lOOkDa filtrate, an S10Y100 (Amicon) ultrafiltration cartridge is used in the SP20 system. The conditioned medium microfiltrate is ultrafiltered according to standard operating procedures. The retentate containing material >100kDa is reduced to approximately 2-10% of the original volume of the conditioned medium microfiltrate and is washed 2X with 2- 10 volumes of 0.4M NaCl . After each washing, the retentate is reduced to its original volume in the lOOkDa ultrafiltration system. After each washing, the permeate is pooled with the previously collected material containing products with molecular weights ≤lOOkDa.

B. Production of lO-lOOkDa cut. To obtain the 10-lOOkDa cut, two S10Y10 ultrafiltration cartridges

(Amicon) are used in the SP20 system. The ≤lOOkDa filtrate is recirculated through the lOkDa ultrafiltration system and the retentate concentrated to 2-10% of the volume of the lOOkDa permeate. The retentate is washed 1-2 times with two to three volumes of Final Buffer (0.1M potassium phosphate, pH 7, 0.1% trehalose and 0.1% glycerol) . After each washing, the retentate is concentrated to its original volume in the lOkDa ultrafiltration system. The 10-lOOkDa concentrate is drained into a sterile vessel and the ultrafiltration cartridges and reservoir are rinsed with a total of 500m-£ Final Buffer, and the rinse is combined with the concentrate.

EXAMPLE 7

Sterile Filtration of Immunomodulator Product In this step, the Immunomodulator product produced in accordance with Example 5 or Example 6 is sterile-filtered through a combination 0.8μm and 0.2μm membrane filter.

EXAMPLE 8 Storage Stability of Immunomodulator Product

Initial activity of Immunomodulator product is determined by adaptation of known lymphoblast proliferation bioassay procedures. See, e . g. , G. Kromer et al., J. Immunol Methods 73, 273-281 (1984) . All stability samples were analyzed by this procedure.

A sample of sterile immunomodulator product produced in accordance with Example 7 above (using material from the large scale procedure of Example 6) was stored at room temperature, and found to retain essentially all activity after a storage period of 40 weeks.

A sample of sterile immunomodulator product produced in accordance with Example 7 above (using material from the large scale procedure of Example 6) was stored under refrigerated conditions at 4°C, and found to retain essentially all activity after a storage period of greater than one year.

A sample of sterile immunomodulator product produced in accordance with Example 7 above (using material from the large scale procedure of Example 6) was stored under frozen conditions at minus 20°C (i.e., a household freezer) , and found to retain essentially all activity after a storage period of 28 weeks.

A sample of sterile immunomodulator product produced in accordance with Example 7 above (using material from the large scale procedure of Example 6) was stored under frozen conditions at minus 70°C, and found to retain essentially all activity after a storage period of over two years. The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. An avian immunostimulant product produced from avian spleen cell conditioned medium, with said product essentially free of proteins having a molecular weight greater than 100 kilodaltons.

2. An avian immunostimulant product according to claim 1, said product essentially free of proteins having a molecular weight less than 10 kilodaltons.

3. An avian immunostimulant product according to claim 1, said product consisting essentially of the avian spleen cell conditioned medium proteins having molecular weights between 10 and 100 kilodaltons.

4. An avian immunostimulant product according to claim 1, wherein said product is a sterile product.

5. An avian immunostimulant product according to claim 1, wherein said product is an aqueous product.

6. An avian immunostimulant product according to claim 1, wherein said product is a frozen aqueous product.

7. An avian immunostimulant product according to claim 1, wherein said product is a lyophilized product.

8. An avian immunostimulant product according to claim 1, wherein said product has a shelf life of at least 3 months at room temperature.

9. An avian immunostimulant product according to claim 1, wherein said product has a shelf life of at least one year under refrigerated conditions.

10. An avian immunostimulant product according to claim 1, wherein said avian spleen cell conditioned medium is a chicken spleen cell conditioned medium.

11. A method of making a sterile concentrated avian immunostimulant product containing Interleukin 2, said method comprising: providing a plurality of avian spleens; then mincing said plurality of avian spleens in an aqueous solution to provide a minced spleen slurry; then homogenizing said minced spleen slurry to provide a crude dissociated spleen cell suspension; then separating tissue and red blood cells from said crude dissociated spleen cell suspension to provide a purified dissociated spleen cell suspension; then incubating said purified dissociated spleen cell suspension in a culture medium in a tissue culture vessel to produce a spleen cell conditioned medium; then removing cells from said conditioned medium to provide an essentially cell free conditioned medium; then separating proteins having a molecular weight greater than 100 kilodaltons from said essentially cell free conditioned medium to produce a dilute immunostimulant product; then separating proteins having a molecular less than 10 kilodaltons from said dilute immunostimulant product to produce a concentrated avian immunostimulant product; and then sterile filtering said concentrated avian immunostimulant product to provide a sterile concentrated avian immunostimulant product.

12. A method according to claim 11, wherein said spleen cells are capsulated spleen cells.

13. A method according to claim 11, wherein said avian spleen cells are chicken spleen cells.

14. A method according to claim 11, wherein said culture medium contains a mitogen.

15. A method according to claim 11, wherein said removing cells step is carried out by continuous flow centrifugation.

16. A method according to claim 11, further comprising, following said removing cells step and prior to said first separating step, the step of: removing particles of a size greater than .5 microns from said spleen cell conditioned medium.

17. An immunostimulant product produced by the method of claim 11.

18. A method of making a crude dissociated avian spleen cell suspension useful in the manufacture of an avian immunostimulant product containing Interleukin 2, said method comprising: providing a plurality of avian spleens; then mincing said plurality of avian spleens in an aqueous solution to provide a minced spleen slurry; then homogenizing said minced spleen slurry to provide a crude dissociated spleen cell suspension; and wherein said homogenizing step is a dispersion homogenization step.

19. A method according to claim 18, wherein said avian spleens are capsulated avian spleens.

20. A method according to claim 18, wherein said avian spleens are chicken spleens.

21. A method of making an avian spleen cell conditioned medium useful in the manufacture of an avian immunostimulant product, said method comprising: providing a dissociated avian spleen cell suspension; incubating said dissociated spleen cell suspension in a culture medium in a tissue culture vessel to produce a spleen cell conditioned medium; said tissue culture vessel including a wall that is impermeable to liquid, permeable to oxygen and permeable to carbon dioxide.

22. A method according to claim 21, wherein said tissue culture vessel comprises a flexible plastic bag.

23. A method according to claim 21, wherein said incubating step is carried out in an atmosphere of from 6.5 to 8.5 percent carbon dioxide.

24. A method according to claim 21, wherein said incubating step is carried out at a temperature of from 37°C to 43°C.

25. A method according to claim 21, wherein said incubating step is carried out at a temperature of from 41°C to 43°C.

26. A method of ultrafiltering an avian spleen cell conditioned medium useful in the production of an avian immunostimulant product, comprising: providing an essentially cell free avian spleen cell conditioned medium; separating proteins having a molecular weight greater than 100 kilodaltons from said conditioned medium to produce a dilute immunostimulant product; and then separating proteins having a molecular less than 10 kilodaltons from said dilute immunostimulant product to produce a concentrated immunostimulant product .

27. A method according to claim 26, wherein said avian spleen cell conditioned medium is a chicken spleen cell conditioned medium.

28. A concentrated immunostimulant product produced by the method of claim 26.