NOVEL METHODS FOR THE PRODUCTION OF PHARMACEUTICAL AGENTS

Abstract

The invention relates a method for producing a pharmaceutical composition, comprising the steps of: providing a liquid collected from a mammal, which liquid comprises cellular constituents of blood, providing a container having an internal surface, contacting said liquid with said internal surface, incubating said liquid contacted with said internal surface for an incubation time, wherein said liquid is agitated with an agitation means at least once during said incubation time, and after said incubation time has passed, obtaining said pharmaceutical composition by steps comprising (1) collecting said liquid or (2) removing part of or the entirety of said cellular constituents from said liquid and collecting the remainder.

Claims

1. A method for producing a pharmaceutical composition, comprising the steps of: (a) providing a liquid collected from a mammal, which liquid comprises cellular constituents of blood, (b) providing a container having an internal surface, (c) contacting said liquid with said internal surface, (d) incubating said liquid contacted with said internal surface for an incubation time, wherein said liquid is agitated with an agitation means at least once during said incubation time, and (e) after said incubation time has passed, obtaining said pharmaceutical composition by steps comprising (1) collecting said liquid or (2) removing part of or the entirety of said cellular constituents from said liquid and collecting the remainder.

2. The method according to claim 1, wherein said container: contains particles for contacting said liquid, which are one or more selected from macroscopic particles, microscopic particles and nanoparticles, has an internal surface that comprises a surface made of one or more selected from glass, plastic, corundum and quartz, or has a volume of 1 ml to 1000 ml.

3. The method according to any of the preceding claims, wherein said liquid is a blood sample, preferably a whole blood sample or a whole blood sample from which erythrocytes have been depleted.

4. The method according to any of the preceding claims, wherein said liquid is (1) agitated during the entirety of said incubation time, (2) agitated two or more times during said incubation time, (3) agitated for a total of at least 5 minutes, (4) agitated at a point in time that is later than 10 minutes after the beginning of said incubation time, (5) not agitated during the first 10 minutes of the incubation time, or (6) agitated by one or more selected from: rotating the container around an axis thereof, moving an axis of the container along a closed trajectory, shifting the container, tilting the container, inverting the container, shaking the container, rotating the liquid, shifting the liquid and stirring the liquid.

5. The method according to any of the preceding claims, wherein said agitation means is selected from a wheel-shaped or propeller-shaped device for rotating the container around an axis thereof, wherein preferably the device has a rotation axis that is perpendicular to the direction of gravity or tilted by an angle of up to 90 degrees, and a shaking device, which is preferably adapted to carry out a circular or translational motion.

6. The method according to any of the preceding claims, wherein said incubation is performed under one or more of the following conditions: for an incubation time of 15 min to 24 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1.5 hours to 24 hours, 2 hours to less than 24 hours, 2 hours to 5 hours, at least 3 hours, 3 hours to 5 hours, 3 hours to less than 24 hours, 3 hours to 20 hours, 3 hours to 16 hours, 4 hours to 12 hours, at least 5 hours, 5 hours to 10 hours, 5 hours to 7 hours, up to 6 hours, at least 6 hours or at least 7 hours, at a temperature from 30° C. to 40° C., preferably at 37° C., or in the absence or presence of an added anticoagulant.

7. The method according to any of the preceding claims, wherein in said pharmaceutical composition the concentration of exosomes is higher than in the absence of agitation under otherwise identical conditions, preferably at least 1.1-fold as high, the concentration of gelsolin is higher than in the absence of agitation under otherwise identical conditions, preferably at least 1.1-fold as high, the concentration of resolvin is higher than in the absence of agitation under otherwise identical conditions, preferably at least 1.1-fold as high the concentration of IL-1Ra is higher than in the absence of agitation under otherwise identical conditions, preferably at least 1.1-fold as high, at least one ratio of the concentrations of an anti-inflammatory mediator (preferably IL-1Ra) and an inflammatory mediator (preferably IL-1β) is higher than in the absence of agitation under otherwise identical conditions, preferably at least 1.1-fold as high, or at least one concentration of an inflammatory mediator (preferably IL-5 or IFN-γ) is essentially the same as before said incubation.

8. The method according to any of the preceding claims, wherein said cellular constituents of blood comprise platelets and step (e) is a step of, after said incubation time has passed, obtaining said pharmaceutical composition by degranulating said platelets by adding calcium ions and/or thrombin, and thereafter collecting said liquid.

9. A pharmaceutical composition produced by the method of any of the preceding claims.

10. A method for producing exosomes, comprising producing a pharmaceutical composition by the method according to any of claims 1 to 8 and collecting exosomes contained in the pharmaceutical composition.

11. Exosomes produced by the method of claim 10.

12. A method for producing an exosome-free preparation, comprising producing a pharmaceutical composition by the method according to any of claims 1 to 8 and removing the exosomes contained in the pharmaceutical composition from the pharmaceutical composition, thereby obtaining said exosome-free preparation.

13. An exosome-free preparation produced by the method of claim 12.

14. The pharmaceutical composition according to claim 9, the exosomes according to claim 11 or the exosome-free preparation according to claim 13 for use as a medicament, preferably by application to the same mammal from which said liquid has been collected.

15. The pharmaceutical composition according to claim 9, the exosomes according to claim 11 or the exosome-free preparation according to claim 13 for use in the treatment of a condition selected from an orthopaedic condition, a joint condition, a tendon condition, pain, a degenerative spinal disease, a condition of an intervertebral disc, a neuro-orthopaedic condition, a condition of a nerve root, irritation of the nervous system, inflammation of the nervous system, a neuropathic condition, a disease involving the immune system, an autoimmune disease, psoriasis, chronic wounds, a muscle disease, food intolerance, drug intolerance, endometriosis, a condition in which apoptosis plays a role, ageing and an ageing-related disorder, for use as an anti-ageing agent or for use as a medicament by injection into the skin.

16. The pharmaceutical composition for use according to claim 14 or 15, the exosomes for use according to claim 14 or 15 or the exosome-free preparation for use according to claim 14 or 15, wherein the use is in combination with one or more other effective agents.

17. Use of the pharmaceutical composition according to claim 9, the exosomes according to claim 11 or the exosome-free preparation according to claim 13 as a cosmetic product.

18. A method for producing a combination, comprising producing (i) a pharmaceutical composition by the method according to any of claims 1 to 8, (ii) exosomes by the method of claim 10 or (iii) an exosome-free preparation by the method of claim 12, and combining said pharmaceutical composition, exosomes and exosome-free preparation, respectively, with a means for storage thereof, a means for application thereof to a mammal or an excipient.

19. A combination produced by the method of claim 18.

20. A kit comprising: a container having an internal surface for being contacted with a liquid collected from a mammal, which liquid comprises cellular constituents of blood, an agitation means for agitating said liquid at least once during an incubation time in which said liquid is incubated in contact with said internal surface and preferably a means for application of a pharmaceutical composition to a mammal, wherein said pharmaceutical composition is obtained by removing part of or the entirety of said cellular constituents from said liquid and collecting the remainder after said incubation time.

21. Use of a container or an agitation means in the method of any of claims 1 to 8.

Description

[0102] FIG. 1 shows the time course of the concentrations of IL-1β and IL-1Ra under two different conditions: static incubation and incubation with slow rotation (x-axis: time of incubation in minutes; y-axis: concentration of IL-1β and IL-1Ra, respectively, in pg/ml).

[0103] FIG. 2 shows the time course of the concentration ratio of IL-1Ra to IL-1β under three different conditions (x-axis: time of incubation in hours; y-axis: ratio of the concentrations of IL-1Ra and IL-1β, both in pg/ml).

[0104] FIG. 3 shows the time course of the concentration of IL-1Ra under three different conditions (x-axis: time of incubation in hours; y-axis: concentration of IL-1Ra in pg/ml).

[0105] FIG. 4 shows the time course of the concentration of IL-1β under three different conditions (x-axis: time of incubation in hours; y-axis: concentration of IL-1β in pg/ml).

[0106] FIG. 5 shows the time course of the concentration of resolvin D1 under three different conditions (x-axis: time of incubation in hours; y-axis: concentration of resolvin D1 in pg/ml).

[0107] FIG. 6 shows the time course of the concentration of exosomes under three different conditions (x-axis: time of incubation in hours, wherein T0 designates the absence of incubation; y-axis: concentration of exosomes, expressed as exosome count per ml).

[0108] FIG. 7 shows the time course of the concentration of gelsolin under three different conditions (x-axis: time of incubation in hours, wherein T0 designates the absence of incubation; y-axis: concentration of gelsolin in ng/ml).

[0109] FIG. 8 shows the time course of the concentration ratio of IL-1Ra to IL-1β under three different conditions (x-axis: time of incubation in hours; y-axis: ratio of the concentrations of IL-1Ra and IL-1β, both in pg/ml).

[0110] FIG. 9 shows the time course of the concentration of IL-1Ra under three different conditions (x-axis: time of incubation in hours; y-axis: concentration of IL-1Ra in pg/ml).

[0111] FIG. 10 shows the time course of the concentration of IL-1β under three different conditions (x-axis: time of incubation in hours; y-axis: concentration of IL-1β in pg/ml).

EXAMPLES

[0112] The following Examples show that according to the present invention, pharmaceutical agents can be produced that are superior as compared to the prior art in various respects.

Example 1: Differential Production of IL-1β and IL-1Ra Due to Agitation During Incubation, One Test Subject

[0113] A pharmaceutical agent was produced by the method of the present invention as follows:

[0114] Peripheral whole blood was collected from a healthy male human being. Blood was drawn into pyrogen-free 10 ml plastic syringes (interior length: ca. 84.5 mm, interior diameter: ca. 14.3 mm, interior surface: ca. 4100 mm.sup.2, interior volume: ca. 13500 mm.sup.3) which were untreated except that they contained purified polished borosilicate glass beads having a diameter of 3.5 mm (EOTII syringes, Orthogen AG, Düsseldorf, Germany).

[0115] The syringes containing the samples were incubated for various incubation times at 37° C. in a Sanyo MCO-20AIC incubator (Japan) without addition of CO.sub.2.

[0116] One part of the samples was incubated without agitation (static incubation).

[0117] Another part of the samples was agitated during incubation. Agitation was achieved by a propeller shaped device for rotating the container around an axis thereof and simultaneously moving an axis of the container along a circular trajectory (Thermo Scientific Tube Revolver, catalogue number 88881001, Thermo Fisher Scientific, Germany), which was set to 15 rpm.

[0118] The device has a rotation axis that was aligned perpendicularly to the direction of gravity. Thus, containers were rotated around their longitudinal axis at 15 rpm, combined with moving the longitudinal axis of the containers along a circular trajectory at an identical frequency. Herein, this motion is also referred to as “slow rotation”.

[0119] After incubation, insoluble components were removed by centrifugation. The resulting conditioned serum was collected and examined by ELISA for IL-1Ra and IL-1β concentrations.

[0120] The results are shown in FIG. 1. Four curves are depicted, as explained in Table 1:

TABLE-US-00001 TABLE 1 Curves shown in FIG. 1 Static incubation Incubation with slow rotation IL-1Ra diamond symbols, square symbols, thick continuous line thick dotted line IL-1β triangle symbols, X-shaped symbols, thin continuous line thin dotted line

[0121] It can be seen that the concentrations of IL-1β remained negligible both when the incubation was static and when the incubation was performed with slow rotation. However, slow rotation led to a pronounced increase in IL-1Ra concentrations over time, as compared to static incubation. Therefore, agitation, in particular slow rotation, improves the level of the desired factor IL-1Ra without affecting the level of the undesired factor IL-1β.

Example 2: Differential Production of IL-1β and IL-1Ra Due to Agitation During Incubation, Four Test Subjects

[0122] As mentioned above, IL-1 (e.g. IL-1β) is a mediator of inflammation, pain and tissue destruction. IL-1Ra is a naturally occurring inhibitor thereof and an important anti-inflammatory cytokine.

[0123] A pharmaceutical agent was produced by the method of the present invention as follows: Peripheral whole blood was collected from four healthy human beings. Blood was drawn into glass beads-containing syringes as described in Example 1.

[0124] The syringes containing the samples were incubated for various incubation times at 37° C. in a Sanyo MCO-20AIC incubator (Japan) without addition of CO.sub.2.

[0125] One part of the samples was incubated without agitation (static incubation).

[0126] Another part of the samples was agitated during incubation with a shaking device carrying out a circular motion with a diameter of 3 mm (IKA MTS 4 shaker, IKA-Werke, Staufen, Germany) at 100 rpm. The syringes were fixed in a horizontal position in plastic plates attached to the shaker. Shaking was done in a horizontal plane. Thus, an axis of the container perpendicular to its longitudinal axis was moved along as circular trajectory at 100 repetitions per minute. The diameter of the closed circular trajectory (the diameter of the respective circle) was therefore 0.3 cm. Herein, this motion is also referred to as “fast shaking”.

[0127] Yet another part of the samples was agitated during incubation by slow rotation as defined in Example 1.

[0128] After incubation, insoluble components were removed by centrifugation. The resulting conditioned serum was collected and examined by ELISA for IL-1Ra and IL-1β concentrations.

[0129] FIG. 2 shows the average ratio of IL-1Ra to IL-1β, FIG. 3 the average concentration of IL-1Ra and FIG. 4 the average concentration of IL-1β. The curves are: static incubation (triangle symbols, dotted line), incubation with fast shaking (square symbols, dashed line), incubation with slow rotation (diamond symbols, continuous line).

[0130] It can be seen that agitation leads to an increase in the concentration of IL-1Ra. In contrast, the concentration of IL-1β was about the same with and without agitation, or even lower in the case of slow rotation. Agitation is thus able to lead to an increase in the ratio of IL-1Ra to IL-1β, which is particularly pronounced in the case of slow rotation. It needs to be kept in mind that the calculated ratios are more prone to error if the IL-1β levels are very low, because such concentrations can be measured less accurately.

[0131] Table 2 indicates the measured ratios of IL-1Ra to IL-1β, Table 3 the measured concentrations of IL-1Ra and Table 4 the measured concentrations of IL-1β. Incubation was performed as static incubation, with fast shaking or with slow rotation as defined above.

TABLE-US-00002 TABLE 2 Ratios of the IL-1Ra concentration to the IL-1β concentration measured after the indicated incubation times (see FIG. 2). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 102.3 582.4 793.2 808.0 274.4 262.4 Fast shaking 692.9 769.2 658.8 141.5 105.3 Static incubation 627.8 658.9 571.0 123.7  80.0

TABLE-US-00003 TABLE 3 IL-1Ra concentrations in pg/ml measured after the indicated incubation times (see FIG. 3). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 61.9 313.3 425.9 758.6 1064.1 1746.1 Fast shaking 350.7 436.0 747.8 1133.7 1288.7 Static incubation 313.9 353.5 590.1  827.8 1186.5

TABLE-US-00004 TABLE 4 IL-1β concentrations in pg/ml measured after the indicated incubation times (see FIG. 4). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 0.6 0.6 0.6 1.8 4.9  9.6 Fast shaking 0.5 0.6 2.2 8.5 15.5 Static incubation 0.5 0.6 1.2 7.4 15.4

[0132] To sum up, it was confirmed that agitation improves the level of the desired factor IL-1Ra without adversely affecting, or favourably affecting, the level of the undesired factor IL-1β. As apparent in particular from the concentration ratio of IL-1Ra to IL-1β, according to the present invention pharmaceutical agents may be produced are even more valuable as an anti-inflammatory agent than those produced using static incubation according to the prior art.

Example 3: Increased Production of Resolvin D1 Due to Various Modes of Agitation

[0133] Certain metabolites (oxylipins) derived from eicosapentaenoic acid (20:5(n-3), also referred to as “EPA”), docosapentaenoic acid (22:5(n-3)) and especially docosahexaenoic acid (22:6(n-3), also referred to as “DHA”) and termed the (neuro)protectins, resolvins and maresins have potent anti-inflammatory and immunoregulatory actions at concentrations in the nanomolar and picomolar range. Resolvin D1, which is a mediator of self-limited resolution of inflammation, was evaluated as an example of such biologically active polyunsaturated fatty acid metabolites. It leads to a suppression of TNF-α expression. Resolvins are also potent analgesics for arthritic and neuropathic pain.

[0134] A pharmaceutical agent was produced by the method of the present invention as follows:

[0135] Peripheral whole blood was collected from four healthy human beings. Blood was drawn into glass beads-containing syringes as described in Example 1.

[0136] The syringes containing the samples were incubated at 37° C. in a Sanyo MCO-20AIC incubator (Japan) without addition of CO.sub.2.

[0137] One part of the samples was incubated without agitation (static incubation).

[0138] Another part of the samples was agitated during incubation by fast shaking as defined in Example 2.

[0139] Yet another part of the samples was agitated during incubation by slow rotation as defined in Example 1.

[0140] After incubation, insoluble components were removed by centrifugation. The resulting conditioned serum was collected and examined by ELISA for the concentration of various factors, including resolvin D1.

[0141] FIG. 5 shows the average concentrations of resolvin D1 over time. The following curves are depicted: static incubation (triangle symbols, dotted line), incubation with fast shaking (square symbols, dashed line), incubation with slow rotation (diamond symbols, continuous line).

[0142] It can be seen that incubation led to a time-dependent concentration increase. Agitation during incubation leads to a more pronounced increase than static incubation. The effect was even stronger with slow rotation than when the sample was agitated by fast shaking.

[0143] Table 5 indicates the measured concentrations. Incubation was performed as static incubation, with fast shaking or with slow rotation as defined above.

TABLE-US-00005 TABLE 5 Resolvin D1 concentrations in pg/ml measured after the indicated incubation times. The absence of incubation is designated referred to as 0 h. 0 h 1 h 2 h 3 h 5 h 6 h 7 h 24 h Slow rotation 33.0 98.9 144.0 188.5 212.3 279.8 275.0 602.2 Fast shaking 94.7 125.9 161.6 209.8 226.9 238.8 516.4 Static incubation 87.8 127.8 133.1 170.7 175.6 217.6 322.4

[0144] The concentrations of IL-5 and IFN-γ were below the threshold of detection.

[0145] Consequently, agitation is able to lead to pharmaceutical agents that may be more efficacious in the resolution of inflammation, suppression of TNF-α expression and analgesia.

Example 4: Increased Production of Exosomes Due to Various Modes of Agitation

[0146] As mentioned above, exosomes are known in the treatment of a variety of conditions. Therefore, the therapeutic value of pharmaceutical agents which contains exosomes may be expected to be dependent on the concentration of exosomes.

[0147] In the same experiment as described in Example 3, also the concentration of exosomes was examined by ELISA.

[0148] FIG. 6 shows the average concentrations of exosomes over time. The following curves are depicted: static incubation (triangle symbols, dotted line), incubation with fast shaking (square symbols, dashed line), incubation with slow rotation (diamond symbols, continuous line).

[0149] It is apparent that incubation led to a time-dependent exosome concentration increase. Agitation is able to increase the concentration of exosomes even further.

[0150] Table 6 indicates the measured concentrations. Incubation was performed as static incubation, with fast shaking or with slow rotation as defined above.

TABLE-US-00006 TABLE 6 Exosome concentrations in exosomes/ml measured after the indicated incubation times. The absence of incubation is referred to as 0 h (T0 in FIG. 6). 0 h 1 h 2 h  3 h Slow rotation 1.164E+11  9.94E+10 2.867E+11 1.2864E+12 Fast shaking 1.281E+11 5.399E+11 1.1099E+12 Static incubation 1.391E+11 5.509E+11  5.553E+11 5 h 6 h 7 h 24 h Slow rotation 1.4008E+12 1.9829E+12 6.9129E+12 1.32379E+13 Fast shaking 1.7237E+12 2.9724E+12 4.0241E+12  4.1858E+12 Static incubation 1.8395E+12 2.8878E+12 2.0937E+12  2.3345E+12

[0151] This means that according to the present invention pharmaceutical agents may be produced that have increased in value in the treatment of the above mentioned conditions.

Example 5: Increased Production of Gelsolin Due to Various Modes of Agitation

[0152] Gelsolin can inhibit apoptosis by stabilising mitochondria. More specifically, it inhibits the release of cytochrome C and thus impedes a signal amplification cascade leading to apoptosis. It is further an actin-binding (capping) protein that also aids in actin polymerisation.

[0153] In the same experiment as described in Example 3, also the concentration of gelsolin was examined by ELISA.

[0154] FIG. 7 shows the average concentrations of gelsolin over time. The following curves are depicted: static incubation (triangle symbols, dotted line), incubation with fast shaking (square symbols, dashed line), incubation with slow rotation (diamond symbols, continuous line).

[0155] It was found that incubation led to a time-dependent increase of gelsolin concentration. Agitation is able to increase gelsolin concentration even further.

[0156] Table 7 indicates the measured concentrations. Incubation was performed as static incubation, with fast shaking or with slow rotation as defined above.

TABLE-US-00007 TABLE 7 Gelsolin concentrations in ng/ml measured after the indicated incubation times. The absence of incubation is referred to as 0 h (T0 in FIG. 7). 0 h 1 h 2 h 3 h 5 h 6 h 7 h 24 h Slow rotation 144.8 240.5 253.7 305.3 390.9 391.2 414.2 415.2 Fast shaking 253.5 254.7 281.1 341.5 293.8 310.9 333.0 Static incubation 262.3 259.3 273.2 280.5 272.5 289.9 302.0

[0157] Consequently, the present invention allows the production of pharmaceutical agents with increased efficacy in the inhibition of apoptosis. This has implications for conditions in which apoptosis plays a role.

Example 6: Differential Production of IL-1Ra Due to Agitation During Incubation, Three Test Subjects

[0158] A pharmaceutical agent was produced by the method of the present invention as follows:

[0159] Peripheral whole blood was collected from four healthy human beings. Blood was drawn into pyrogen-free 10 ml plastic syringes (interior length: ca. 84.5 mm, interior diameter: ca. 14.3 mm, interior surface: ca. 4100 mm.sup.2, interior volume: ca. 13500 mm.sup.3), which neither contained glass beads nor contained any other particles for contacting the liquid.

[0160] The syringes containing the samples were incubated for various incubation times at 37° C. in a Sanyo MCO-20AIC incubator (Japan) without addition of CO.sub.2.

[0161] One part of the samples was incubated without agitation (static incubation).

[0162] Another part of the samples was agitated during incubation by fast shaking as defined in Example 2.

[0163] Yet another part of the samples was agitated during incubation by slow rotation as defined in Example 1.

[0164] After incubation, insoluble components were removed by centrifugation. The resulting conditioned serum was collected and examined by ELISA for the concentrations of IL-1Ra.

[0165] FIG. 8 shows the average ratio of IL-1Ra to IL-1β, FIG. 9 the average concentration of IL-1Ra and FIG. 10 the average concentration of IL-1β. The curves are: static incubation (triangle symbols, dotted line), incubation with fast shaking (square symbols, dashed line), incubation with slow rotation (diamond symbols, continuous line).

[0166] It can be seen that also in the absence of particles for contacting the liquid, agitation leads to an increase in the concentration of IL-1Ra.

[0167] In contrast, the concentration of IL-1β was about the same with and without agitation, or even lower in the case of slow rotation. Also in the absence of particles, and potentially depending on incubation time, agitation is thus able to lead to an increase in the ratio of IL-1Ra to IL-1β, which may be particularly pronounced in the case of slow rotation. It needs to be kept in mind that the calculated ratios are more prone to error if the IL-1β levels are very low, because such concentrations can be measured less accurately.

[0168] Table 8 indicates the measured ratios of IL-1Ra to IL-1β, Table 3 the measured concentrations of IL-1Ra and Table 4 the measured concentrations of IL-1β. Incubation was performed as static incubation, with fast shaking or with slow rotation as defined above.

TABLE-US-00008 TABLE 8 Ratios of the IL-1Ra concentration to the IL-1β concentration measured after the indicated incubation times (see FIG. 8). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 633.9 1529.6 2022.0 2091.0 2426.2 4244.3 Fast shaking 1725.1 1906.2 2367.5 2340.9  930.2 Static incubation 1428.1 1442.5 1560.2 2989.2  904.1

TABLE-US-00009 TABLE 9 IL-1Ra concentrations in pg/ml measured after the indicated incubation times (see FIG. 9). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 126.8 305.9 404.4 418.2 485.2 1236.1 Fast shaking 345.0 381.2 473.5 738.9 1050.8 Static incubation 285.6 288.5 312.0 673.9  700.3

TABLE-US-00010 TABLE 10 IL-1β concentrations in pg/ml measured after the indicated incubation times (see FIG. 10). 0 h 1 h 2 h 3 h 5 h 6 h Slow rotation 0.2 0.2 0.2 0.2 0.2  3.3 Fast shaking 0.2 0.2 0.2 1.4  9.6 Static incubation 0.2 0.2 0.2 0.2 12.4

[0169] Agitation improves the level of the desired factor IL-1Ra also in the absence of glass beads or other particles. As compared with their presence, the levels of IL-1Ra are somewhat lower, consistent with the explanation that such particles increase the surface for contacting the liquid and improve induction. Excellent ratios of IL-1Ra to IL-1β and low levels of IL-1β may also be achieved in the absence of particles.