GELATIN PURIFICATION

Abstract

Described is an improved method of removing lipopolysaccharide from an aqueous medium comprising gelatin and lipopolysaccharides, said method comprising the steps of providing an aqueous medium comprising gelatin and lipopolysaccharides, adding to the aqueous medium a micelle-forming surfactant, contacting the medium with a solid adsorbent, separating the solid adsorbent of from the medium and recovering the aqueous medium comprising the gelatin, wherein the method is performed at conditions below the cloud point of the surfactant.

Claims

1. Method of removing lipopolysaccharide from an aqueous medium comprising gelatin and lipopolysaccharides, said method comprising the steps of: 1) providing an aqueous medium comprising at least 2 w/w % gelatin and lipopolysaccharides, 2) adding to the aqueous medium 0.01-1.5 w/w % of a micelle-forming surfactant, 3) contacting the medium of step 2) with a solid adsorbent, 4) separating the solid adsorbent of step 3) from the medium, 5) recovering the aqueous medium comprising the gelatin, wherein each of the steps 1)-5) are performed at a temperature of 68° C. or less, said temperature being below the cloud point of the micelle-forming surfactant, at least steps 2) and 3) being performed at a temperature of at least 30° C.

2. Method of claim 1, wherein the average molecular weight of the gelatin is between 1500 Da and 250,000 Da, preferably between 2000 and 200,000 Da, more preferably between 5000 and 180,000 Da, most preferably between 20,000 Da and 170,000 Da.

3. Method of claim 1, wherein the micelle-forming surfactant comprises a non-ionic surfactant.

4. Method of claim 3, wherein the non-ionic surfactant is an ethoxylated surfactant, preferably an alkylphenol ethoxylate.

5. Method of claim 1, wherein the alkylphenol ethoxylate is represented by the formula CxH2x-1-C6H4-O—(C2H4O)nH, wherein x is 4-12 and n is 7.5-14.

6. Method of claim 4 wherein x is 8 and n is 8-13, preferably 8.5-12.5, most preferably 9-12.

7. Method of claim 1, wherein the surfactant is Triton X-100 or Triton X-102, or mixtures thereof

8. Method of claim 1, wherein the solid adsorbent is a hydrophobic adsorbent.

9. Method of claim 8, wherein the hydrophobic adsorbent comprises activated carbon.

10. Method of claim 1, wherein each of the steps 1)-5) are performed at a temperature of 65° C. or less, preferably of 62° C. or less most preferably of 60° C. or less.

11. Method of claim 1, wherein at least steps 2) and 3) are performed at a temperature of at least 35° C., preferably at least 40° C., more preferably at least 45° C., most preferably at least 50° C.

12. Method of claim 1, wherein each of steps 1)-5) are performed at a temperature of at least 30° C., preferably at least 35° C., more preferably at least 40° C., even more preferably at least 45° C., most preferably at least 50° C.

13. Method of claim 1, wherein the pH of the medium is between 3.5 and 9.0, preferably between 4.0 and 8.0, more preferably between 4.0 and 6.0, most preferably between 4.5 and 5.5.

14. Method of claim 1, wherein the aqueous medium in step 1) comprises at least 8 w/w %, preferably at least 12 w/w %, even more preferably at least 20 w/w % dissolved gelatin.

15. Method of claim 1 wherein the aqueous medium comprises up to 37 w/w % dissolved gelatin, preferably up to 30 w/w %.

16. Method of claim 1, wherein in step 2), the weight ratio of gelatin to added surfactant is 2000:1 or less, more preferably 500:1 or less, even more preferably 250:1 or less, most preferably 50:1 or less.

17. Method of claim 16, the weight ratio of gelatin to added micelle forming surfactant is 50-5:1

18. Method of claim 1, wherein in step 2), the surfactant is added to a concentration of 0.015-1.0 w/w %, preferably of 0.02-0.50 w/w %.

19. Method of claim 1, wherein the surfactant is added to above the critical micelle concentration thereof.

20. Method of claim 1, wherein step 2) comprises incubating the medium for at least 1 minute after adding the surfactant.

21. Method of claim 20, wherein the medium is incubated 2 minutes to 1 hour, preferably for 5-30 minutes.

22. Method of claim 1, wherein in step 3), the solid adsorbent is added to the medium in a weight ratio to the surfactant of at least 2.5:1, preferably of at least 3.0:1, more preferably of at least 3.5:1, even more preferably of 0.1-3 w/w % and most preferably of 0.5-1 w/w %.

23. Method of claim 1, wherein steps 3) and 4) comprise passing the medium obtained after step 2) through one or more filter elements comprising the solid adsorbent.

24. Method of claim 23, wherein steps 3), 4) and 5) comprise passing the medium obtained after step 2) through one or more filter elements comprising the solid adsorbent.

25. Method of claim 1, wherein step in 5) comprises filtration, separating the solid adsorbent from the medium.

26. Method of claim 1, wherein the method is free of a centrifugation step.

27. Method of claim 1, wherein the method is free of an ultrafiltration step.

28. Method of claim 1, wherein the aqueous medium has a salt content 100 mM or less, preferably of 80, 70, 60 or 50 mM or less, most preferably of 40, 30 or 20 mM or less during steps 1)-5).

29. Method of claim 1, wherein the aqueous solution is free of acetone and/or alcohol in steps 1)-5).

30. Method of claim 1, further comprising incubating the aqueous medium with an oxidizing agent.

31. Method of claim 30, wherein the oxidizing agent is added during any of step 1), 2) or 3), preferably during step 2).

32. Method of claim 30, wherein the oxidizing agent is chosen from hydrogen peroxide and peracetic acid and mixtures thereof.

33. Method of claim 30, wherein the oxidizing agent is added in a concentration of 0.5-2.5 w/w %.

34. Method of claim 1, wherein the aqueous medium in step 1) has a lipopolysaccharide content of 1500 EU/g dry weight of gelatin or less, preferably 1000 EUg/g dry weight of gelatin.

35. A Gelatin, being substantially free of quaternary ammonium slats, comprising gelatin derived molecules having a molecular weight of above 100,000 Da, most preferably above 120,000 Da, having a lipopolysaccharide content of less than 100 EU/g, more preferably less than 50 EU/g, even more preferably less than 20 EU/g, even more preferably less than 10 EU/g, even more preferably less than 5 EU/g, even more preferably less than 2 EU/g, most preferably less than 1 EU/g.

36. The Gelatin, obtainable by the method of claim 1, having a lipopolysaccharide content of less than 2 EU/g, preferably less than 1 EU/g.

37. The Gelatin being of type A, obtainable by the method of claim 1, comprising gelatin derived molecules having a molecular weight of above 100,000 Da, most preferably above 120,000 Da, having a lipopolysaccharide content of less than 100 EU/g, more preferably less than 50 EU/g, even more preferably less than 20 EU/g, even more preferably less than 10 EU/g, even more preferably less than 5 EU/g, even more preferably less than 2 EU/g, most preferably less than 1 EU/g.

38. The Gelatin of claim 35 wherein the average molecular weight of the gelatin is between 1500 Da and 250,000 Da, preferably between 2000 and 200,000 Da, more preferably between 5000 and 180,000 Da, most preferably between 20,000 Da and 170,000 Da.

39. The Gelatin of claim 35, the gelatin having an average molecular weight of above 80,000 Da, preferably above 100,000 Da, most preferably above 120,000 Da.

40. The Gelatin of claim 35, being free of acetone and/or quaternary ammonium salts/and or alcohols.

41. An Aqueous medium comprising at least 2 w/w % gelatin of claim 35, said medium having a salt content of 100 mM or less, preferably 50 mM or less, most preferably 20 mM or less.

42. The Aqueous medium of claim 41, being substantially free of acetone and/or quaternary ammonium salts.

43. The Aqueous medium of claim 41, comprising at least 6 w/w %. preferably at least 10 w/w %, more preferably at least 15 w/w % and most preferably at least 20 w/w % gelatin.

Description

[0072] The invention will now be further described by way of non-limiting examples and figures.

[0073] FIG. 1 is a graph, shows the effect of surfactant concentration on the surface tension of an aqueous gelation solution at 25° C. The CMC of Triton X-100 is found to be 0.015-0.018, equal to the said CMC in water.

[0074] FIG. 2 is a graph, showing the surface tension of aqueous gelatin solutions as function of the ratio adsorbent to remove Triton X-100 surfactant from the solution.

[0075] FIG. 3 is a graph, showing the effect of the length of the polyoxyethylene moiety in different Triton species used on the purification. The X axis represents the n number in the C.sub.8H.sub.15—C.sub.6H.sub.4—O—(C.sub.2H.sub.4O).sub.nH, and the Y axis shows the LPS content in EU/g in a gelatin containing LPS after purification.

EXAMPLES

[0076] A full overview of the gelatins used in the different examples is listed in table 1.

[0077] The analysis method to determine the gelatin properties is described in GME10.

[0078] In the examples, removal of surfactant was monitored, as surfactant, such as Triton X100 can mask LAL analysis results. See example 4 for further details.

[0079] Unless indicated otherwise, mixing was performed with a speed of 750 rpm using a water-bath mixer from IKA Werke Germany, model R015 power and a standard magnetic stirrer bar of 4-5 cm length.

[0080] Unless otherwise indicated, the weight of gelatin indicated includes a moisture content of 10-13 w/w %.

Example 1

[0081] Endotoxin Purification from Aqueous Gelatin Solutions of Different LPS Content

[0082] 6.66 w/w % gelatin solutions were prepared by weighing 50 g of the gelatin batches 1, 3, 4, 5, 6 and 8, 15, 16 and 17 with different initial LPS contents, varying from ˜1000 to about 34,000 EU/g gelatin, (see table 1.1) with 700 ml water. The gelatins shown in Table 1 have a moisture content of between 10.3 and 12.6 w/w %. So the actual gelatin content on dry matter basis is 87.5-89.7%.

[0083] The mixture was kept at ambient temperature for 30 minutes to allow the gelatin to swell/hydrate. Subsequently, the gelatin was brought in solution by elevation of the temperature to a maximum of 60° C. under constant mixing for 30-45 minutes with a with a speed of 750 rpm. The pH of the gelatin solution was measured to be between 5.2 and 5.6, no further adjustment of pH was made. A sample was taken and the initial LPS content was measured. The Endosafe LAL method, from Charles River USA and the EndoZyme recombinant factor C method from Hyglos GmbH (Germany) were both used to analyse the LPS levels in the gelatins before and after purification.

[0084] Both methods were used according to the instructions of the manufacturer to determine the LPS content.

[0085] For the LPS analysis 1000 mg gelatin was dissolved in 40.0 ml deionized pyrogen free water. The gelatin was completely dissolved by heating the solution to 55° C. for 30-45 minutes, adjusted to 40° C. and appropriately diluted before the LPS analysis was executed.

[0086] Next, 1.4 g (0.18 w/w %) Triton X100 (Carl-Roth, Germany, product number 3051.4) was added to the gelatin solution and the gelatin—Trion X100 solution was, under constant mixing with a speed of 750 rpm, placed at 75° C. for 30 minutes.

INCORPORATED BY REFERENCE (RULE 20.6)

[0087]

TABLE-US-00001 TABLE 1.1 Gelatin starting materials Process Gelatin and Batch raw Endotoxin bloom viscosity Mw Cond. Moisture Production number material EU/g g mPas kDa pH IEP μS/cm % location** 1 Type A 1000-1100 311 4.4 130 5.79 8.65 95 10.7 G pigskin 2 Type A 2800-3400 329 4.9 146.2 5.5 8.7 112 11.6 G pigskin 3 Type A 4900-5100 302 4.22 133 5.44 8.76 115 11.9 G pigskin 4 Type A 4100-4200 305 4.16 129.3 5.47 8.58 81 12 G pigskin 5 Type A 5000-5200 220 4.23 75.1 5.62 8.57 179 11.3 G pigskin 6 Type A 2400-2500 306 4.12 128.4 5.28 8.65 134 11.7 G pigskin 7 Type A 2800-3000 270 5.83 169 4.73 8.65 225 11.9 A pigskin 8 Type A 2800-3000 316 5.2 149.1 5.56 8.71 126 10.9 G pigskin 9 Type A 6800 53 1.6 50.5 5.23 7.64 185 12.6 G pigskin 10 Type A 17000-18000 0 5.0* 4.9 5.2 7.5 434 7.5 A pigskin 11 Type A 32000-34000 275 3.55 104.3 6 8.6 108 12.2 A fish 12 Type B 2900-3200 264 5.32 151.2 5.72 5.06 113 10.4 P bovine bone 13 Type B 6700-6800 254 5.38 153.1 5.74 4.86 102 10.3 P bovine bone 14 Type B 200-300 263 4.08 123 5.74 5.05 127 11.1 I bovine bone 15 Type A 370-465 300 4.9 145.0 5.40 8.8 118 11.0 G pigskin 16 Type A 11000-12000 150 1.8 72 5.50 8.7 133 10.1 A pigskin 17 Type A 10000-11000 150 2.2 78 5.15 8.7 155 11.7 G pigskin *viscosity was measured according to GME10, however, instead of a 6.67 w/w % solution, a 20 w/w % solution was used at 25° C. **G: Rousselot bvba, Gent, Belgium; A: Rousselot AS, Angouleme, France; P: Rousselot Inc., Peabody, USA; I: Rousselot SASIaI, Isle sur la Sorgue, France

[0088] Subsequently, a minimum of 5.0 g active carbon (Norit SX-Plus, Cabot, the Netherlands) was added (0.7 w/w %), followed by an additional 30 minutes mixing (500-1000 rpm) at 60° C. Next, the solution was filtered over a 0.45 μm filter (Phenex RC 26 mm, 0.45 μm (Phenomenex, The Netherlands) to remove active carbon and cooled to 40° C. for direct LPS analysis on the purified solution, or frozen at −20° C. and freeze dried using a Christ Alpha 2-4LD Plus freeze-dryer (MartinChrist, Germany). Freeze-drying vacuum conditions: 0.04 mbar and −87° C. for at least 24-48 hours until the solutions are dried to a moisture content of around 4-6%. No moisture correction was done before endotoxin analysis.

[0089] Filtration of an initial (non-purified) gelatin solution over a Phenex 0.45 μm filter did not influence or reduce the initial LPS level in the gelatin.

[0090] It can be observed from the data from table 1.2 that a very efficient LPS removal can be obtained from the starting materials. In order to obtain gelatins with an endotoxin content as low as 2 EU/g or less, it is preferred to start with a gelatin solution having 1500 EU/g enotoxin or less.

TABLE-US-00002 TABLE 1.2 LPS reduction in different gelatin batches Initial LPS Purified LPS Purification Gelatins level (EU/g) level (EU/g) factor Gelatin 1 1100 1 1100 Gelatin 3 4960 190 26 Gelatin 4 4200 142 30 Gelatin 5 5120 28 182 Gelatin 6 2480 109 23 Gelatin 8 2912 39 75 Gelatin 15 372 1 372 Gelatin 15 465 2 232 Gelatin 16 11467 7 1638 Gelatin 17 10728 9 1192

Example 2

[0091] Variation of Surfactant Concentration

[0092] Three 6.66% w/w gelatin solutions were prepared as described for example 1, using the gelatin batches 1, 2 and 15, having almost similar average molecular weight and viscosity, see table 1. Different amounts of Triton X100 (Carl-Roth, product number 3051.4) were added to the solution (see table 3) followed by mixing at maximum 60° C. for 30 minutes. Subsequently, 5.0 g (0.7 w/w %) active carbon (Norit SX-Plus) was added to tests 2-3-4-5. Followed by mixing at maximum 60° C. for 30 minutes at 500-1000 rpm and removed as described in example 1, using a 0.45 μm filter (Phenex RC 26 mm, 0.45 μm). The Active carbon amounts was increased for test 6 to respectively 20 g to assure that all Triton X100 will be removed from the purified gelatin solution. Surface tension analysis confirmed indeed that the Triton X100 concentration were reduce to value below the CMC.

[0093] After purification the gelatins were stored at −20° C. and freeze-dried as described in example 1. The freeze-dried gelatins were used for the LAL LPS analysis.

[0094] Table 2 indicates that a triton concententration of above the CMC thereof is shown to be advantageous for endotoxin removal.

TABLE-US-00003 TABLE 2 LPS reduction on different levels of Triton X100 Triton x Conc. 100 g Triton LPS value LPS value LPS value per 50 g X100 (EU/g) (EU/g) (EU/g) Test gelatin (w/w %) Gelatin 1 Gelatin 2 Gelatin 15 1 0 0 1100 5400 442 2 0.048 0.0065 1000 3800 3 0.097 0.014 100 450 4 0.194 0.026 16 38 5 1.36 0.18 1-10 16 1 6 7.95 1.05 <1 15 1

Example 3

[0095] Diatomaceous Earth as LPS Purification Agent

[0096] 600 ml 6.66 w/w % gelatine 1 and 9 solution was prepared at pH 5.5, and treated with Triton X100 as described in example 1. After the incubation step of 30 minutes at a maximum temperature of 60° C., instead of active carbon, 70 gram diatomaceous earth (Claracel CBL), pre-washed with deionized water, was added followed by 4 hours continuous mixing at 50° C. After 4 hours, the diatomaceous earth was removed by filtration (Whatman Glass microfiber GF/C grade, 55 mm diameter, 2 mm. Schleicher & Schuell, Germany). The filtered gelatin solution was overnight stored at −20° C., followed by freeze-drying (see example 1 for the freeze-drying conditions). A non-purified 6.67% gelatin 1 and 9 sample were also stored at −20% and freeze-dried. The LPS content was analyzed on the purified and non-purified freeze-dried gelatin samples, see table 3.

[0097] A significant amount of the endotoxin can be removed from the gelatin using diatomaceous earth as adsorbent. The LPS purification is however somewhat less efficient as compared to that when active carbon is used, see gelatin 1. However, it is also possible to apply diatomaceous earth in a pre-purification step.

TABLE-US-00004 TABLE 3 LPS reduction with diatomaceous earth as adsorbent Initial LPS Purified LPS Purification Gelatins level (EU/g) level (EU/g) factor Gelatin 1 1036 39 26 Gelatin 9 6800 300 23

Example 4

[0098] Variation of Amount of Adsorbent

[0099] The efficiency of removal of surfactant by the adsorbent is analyzed by measuring the surface tension of the samples before addition of the surfactant to the medium comprising the gelatin, and compared with the surface tension measured after treatment with adsorbent and removal thereof. Surface tension drops in the presence of surfactant, e.g. Triton X100. FIG. 1 shows that the initial surface tension of a 1 w/w % gelatin solution of 65-67 mN/m drops significantly starting at a Triton concentration of 0.001 w/w %, based on the weight of the solution. The critical micelle concentration of Triton X100 is between 0.014 and 0.018% w/w.

[0100] Surface tension was analyzed using the Digidrop (GBX, France) contact angle/surface tension analysis equipment. The needle diameter was 0.81 mm and the drop formation speed was 0.384 μl/s. Maximum drop volume is 9.900 μl. Surface tension was calculated using the ds/de equation.

[0101] In case high amounts of surfactant are used, a corresponding higher amount of adsorbent may be needed to remove the said surfactant from the solution. It is also possible to repeat the adsorption step in order to remove any residual surfactant, not removed in a first round of adsorption, until a surface tension value of 65-67 mN/m equal to pure gelatin is obtained.

[0102] Solutions of 50, 60 and 100 g gelatin 7 (see table 1) in 700 ml water were prepared as described in example 1, resulting in gelatin concentrations of 6.66, 8.0 and 12.5 w/w %, respectively. Triton X100 (Carl-Roth) amounts added were 1.4 g (0.18 w/w %) for a 6.67% gelatin solution, 1.7 g (0.216 w/w % for a 8% gelatin solution and 2.8 g (0.36 w/w %) for a 12.5% gelatin solution. Mixing was done at a speed of 500-1000 rpm at 60° C.

[0103] The active carbon (Norit SX-Plus) amounts added were varied and also increased in line with the Triton X100 concentration increase, see table 4. After active carbon addition the mixture was mixed for an additional 30 minutes at 60° C. at a speed of 500-1000 rpm. Finally the solutions were filtered equally to example 1 using 0.45 μm filter (Phenex RC 26 mm, 0.45 μm). The filtered solutions were used to measure the surface tension, see table 4. It can be observed from table 4 and FIG. 2 that at a weight ratio active carbon:Triton X100 of 2.5 or higher results in a surface tension close to the initial gelatin solution without added surfactant. At a weight ratio of 3 or higher, in particular of 3.5 or higher, the surface tension are equal to that of the initial gelatin solution, indicating that the surfactant has been removed substantially completely. A higher (above 3.5) active carbon: Triton ratio results in an even more efficient Triton X100 reduction. See also FIG. 2.

TABLE-US-00005 TABLE 4 Active carbon/Triton ratio and effect on surface tension Triton X100 Gelatin g Ratio Surface Active g (w/w % in (w/w % in active carbon: Tension carbon solution) solution) Triton X100 (mN/m) — — 100 g (12.5) — 66.1 5 g 1.35 g (0.18)  50 g (6.7) 3.7 65.8/66.0 6 g 2.0 g (0.26) 60 g (8) 3.0 65.0 8 g 2.7 g (0.34) 100 g (12.5) 3.0 64.5/66.5 1 g 2.7 g (0.34) 100 g (12.5) 0.37 35.36 2 g 2.7 g (0.34) 100 g (12.5) 0.74 35.7 4 g 2.7 g (0.34) 100 g (12.5) 1.48 36.98 6 g 2.7 g (0.34) 100 g (12.5) 2.22 51 8 g 2.7 g (0.34) 100 g (12.5) 3 64 8 g 2.8 g (0.35) 100 g (12.5) 2.9 66.0 10 g  2.7 g (0.34) 100 g (12.5) 3.7 66

Example 5

[0104] Temperature Variation, Influence on LPS Removal and Functionality.

[0105] A same test as described in example 2 was performed on gelatin 1. pH adjustment was done to a value of 5.5. After Triton X100 addition, the solution was mixed at 60° C. for 15 minutes, and subsequently, the temperature was adjusted to the temperatures listed in table 5 followed by an additional maximum 30 minutes mixing at 500-1000 rpm. Two different Triton X100 concentrations were used, 0.18 and 0.026 w/w %.

[0106] Subsequently, treatment with active carbon and solution filtration was performed according to example 1. In addition to LPS analysis, also analysis of the viscosity of the solutions as well as the average molecular weight of the gelatin were performed as an indication of functionality of the gelatin after treatment.

[0107] Viscosity was analyzed according to the method described in the GME10. Molecular weight distribution was measured according to Olijve et. al., supra.

[0108] Gelatins were freeze-dried before LPS analysis as described before.

TABLE-US-00006 TABLE 5 Temperature variation at pH 5.5 - gelatin 1 Triton X100 concentration (w/w %) 0.18 0.026 Temperature Mw viscosity Purified LPS Purified LPS (° C.) (kDa) (mPas) level (EU/g)) level (EU/g) non-treated 130 4.4 1050 1050 57.5 129.5 4.4 2 6 65.0 127.5 4.3 5-6 6 80.0 110 4 8 11 90.0 46 0.8 10 20

[0109] It can clearly be observed that at a temperature of 90° C., the gelatin is hydrolyzed and loses its functionality. The viscosity decreases from an initial value of 4.4 to 0.8 mPas and the average molecular weight decreases from 130 to 46 kDa, i.e. a molecular weight loss of 65%. Also at a temperature of 80° C., the reduction in molecular weight and viscosity is significant as well. However, at temperatures of 65° C. or lower (below the cloud point of Triton X100), where significant hydrolysis is prevented, functionality is maintained, and surprisingly, a very efficient LPS removal is observed, which is equal to, or at lower Triton X100 concentration, even slightly better than at higher temperatures.

Example 6

[0110] pH and Temperature Variation, Influence on LPS Removal and Functionality

[0111] This example was performed as described in example 5. Gelatins 1, 2, 3, 7, 8 and 10 were used for the purification. In addition to the temperature, also pH of the purification solution was adjusted and varied. After addition of Triton X100 (0.026 and 0.18 w/w %), the temperature was adjusted from 57.5 to 90° C. as indicated in table 6.1 and followed by 30 minutes mixing at 500-1000 rpm. Subsequently, 5.0 gram active carbon (Norit SX-Plus) was added and the solution was mixed for an additional 15-30 minutes at 500-1000 rpm. Next the solution was filtered using a 0.45 μm filter as described in the previous examples. Gelatins were freeze-dried before LPS analysis as described before. In table 6.1 temperature variation was executed with gelatin 7 with a pH adjustment to pH 4.5. Temperature will become much more critical in relation to gelatin hydrolysis at lower pH values. Besides the endotoxin (LPS) analysis also the molecular weight and viscosity values were measured after purification to observe possible gelatin hydrolysis and loss in gelatin properties. Viscosity and molecular weight distribution analysis was measured using the methods mentioned in example 5.

TABLE-US-00007 TABLE 6.1 Temperature variation at a pH of 4.5 - gelatin 7 Triton X100 concentration (%) 0.18 0.026 temperature Mw viscosity Purified LPS Purified LPS (° C.) (kDa (mPas) level (EU/g) level (EU/g) non-treated 169 5.8 2900 2900 57.5 169.5 5.8 15 28 65 158.6 5.4 12 21 80 140.0 4.6 15 45 90 52.0 1.2 16 35

[0112] It is observed that a temperature above 65° C., in particular at 80 and 90° C., and a low pH of 4.5 leads to loss of molecular weight and viscosity under the test conditions used, see table 6.1. Therefore, the gelatin is preferably be held at 65° C. or lower during the steps of the method for 15 minutes or less. Most preferably, the temperature during the steps of the method do not exceed 60° C. if the pH is 4.5 or lower, such as 4.0.

[0113] To confirm the results of table 6.1 a wider pH range was tested using gelatin 2, 3 and 7 at temperatures of 57.5° C., i.e. below and above the temperature where gelatin hydrolyses (60° C.).

[0114] The pH ranges applied are listed in the table 6.2. The pH adjustment of the gelatin solution was done before the Triton X100 addition with 0.1M hydrochloric acid (Sigma Aldrich, 258148-500ML) or 0.1M NaOH (Sigma-Aldrich, USA, 221465-500G). Instead of hydro-chloric acid also other acids, such as sulfuric acid (Sigma-Aldrich) can be used to lower the pH. It is to be observed that the chloride concentration after pH adjustment was below 50 mM, which concentration does not affect the cloud point of the surfactant used.

[0115] The gelatin solution preparation and the Triton X100 (0.18 w/w %), active carbon addition (5 gram 0.7 w/w %), mixing and filtration of the gelatin samples was equal to the methods described in the previous examples. Gelatins were freeze-dried before LPS analysis as described before. The LPS purification results at 57.5° C. and the molecular weight and viscosity measured after purification are provided in table 6.2.

[0116] Variation of the pH at 57.5° C. did not result in phase separation of the micellar aqueous phase.

TABLE-US-00008 TABLE 6.2 pH variation at 57.5° C., Triton X100 0.18% (W/W) Gelatin 2 Gelatin 3 Gelatin 7 Purified Purified Purified Mw Viscosity LPS Mw Viscosity LPS Mw Viscosity LPS pH (kDa) (mPas) (EU/g) (kDa) (mPas) (EU/g) (kDa) (mPas) (EU/g) 3 109 3.7 101.3 3.15 127.7 4.2 14 4 130 4.4 118.9 3.85 151.6 5.2 15 4.5 139 4.7 10 129.7 4.15 20 167.5 5.8 15 5 142 4.8 131.6 4.2 5.5 144 4.8 140 133.8 4.3 150 166.9 5.9 45 6 145 4.9 134.6 4.3 168.2 5.92 118 7 145.5 4.9 134.8 4.3 168.5 5.95 140 Non- 146.2 4.9 135.1 4.22 169.4 5.90 2850 treated gelatin

[0117] Variation of pH at 57.5° C. results in limited loss in molecular weight and viscosity at pH values above 4.5, see table 6.2. Significant LPS purification without loss in molecular weight/viscosity is obtained between pH values of 4.5 and 5.6. In particular for gelatin 7.

[0118] Various gelatins (1, 2, 3, 7, 8, 10) were tested at a temperature of 57.5° C. at pH 4.5 and 5.5 to compare purification efficiency without loss in molecular weight and viscosity, table 6.3. The gelatin solution preparation was performed as described above. If required, the pH was adjusted to 4.5 and 5.5 with 0.1M hydrochloric acid (Sigma Aldrich, 258148-500ML) or 0.1M NaOH (Sigma-Aldrich, 221465-500G). Instead of hydrochloric acid also other acids such as sulfuric acid (Sigma-Aldrich) can be used.

[0119] The Triton X100 concentration used was 0.18 w/w % and a minimum of 5 g active carbon was admixed. The gelatin solution was filtered before LPS analysis as described for the previous examples. Gelatins were freeze-dried before LPS analysis as described above.

[0120] An improved LPS purification is visible at pH values of 4.5 compared to 5.5. The influence of improved LPS purification at lower pH looks to be larger at higher initial LPS values. Lower pH is preferred in case low LPS levels (<20 EU/g) are required. No significant change in molecular weight distribution was observed between pH 4.5 and 5.5 for the tested gelatins at 57.5° C., values not listed.

TABLE-US-00009 TABLE 6.3 LPS removal at pH 4.5 and 5.5 at 57.5° C. for different gelatins Initial LPS Purified LPS level Purified LPS level Gelatins (EU/g) (EU/g) at pH 5.5. (EU/g) at pH 4.5. Gelatin 1 1050 2-7 2-5 Gelatin 2 3400 140 10 Gelatin 3 5040 150 20 Gelatin 7 2850 45 15 Gelatin 8 2950 33 15 Gelatin 10 17640 288 55

Example 7

[0121] LPS Removal Above and Below the Cloud Point of the Surfactant

[0122] Purpose was to measure the LPS removal from gelatin solutions at conditions below the cloud point of the surfactant used, as compared to conditions above the cloud point. In order to keep the conditions similar, temperature conditions of 57.5° C. were used while using Triton X-100 (cloud point of 68° C.) as well as Triton X-114 (Sigma-Aldrich, cloud point of 23° C.) as surfactant.

[0123] Gelatin 7 was used to prepare a gelatin solution as described in the previous examples. The pH applied was 4.7. Triton X-100, Triton X-114 or mixtures thereof were used as surfactant in a concentration of 0.18 w/w %. After the surfactant was added to the aqueous gelatin medium, the temperature was adjusted to 57.5° C. followed by 15-30 minutes mixing. Active carbon was added to an amount of at least 5 gram (0.7 w/w %) and an additional mixing was done for 15-30 minutes. After filtration, as described in the previous experiments, the gelatins were freeze-dried before LPS analysis as described above.

[0124] Results are given in table 7. It can be seen that when using Triton X-100 at a temperature above the cloud point thereof, i.e. at a temperature of 75° C., a gelatin solution is obtained that still contains 27 EU/g LPS, as compared to only 15 EU/g when the method was performed at 57.5° C., i.e. below the said cloud point. This means that LPS removal is more efficient when the method is performed below the cloud point of the surfactant. In addition, at 75° C., significant hydrolysis of gelatin occurs, resulting in undesired loss of viscosity and functionality, see e.g. example 6. At 75° C., the viscosity reduced from 5.8 mPas to about 4.9 mPas, whereas at 57.5° C., the viscosity remained 5.8 mPas. Using Triton X-114 at 57.5° C. resulted in a gelatin solution still having 114 to 150 EU/g LPS, indicating that in comparison with Triton X-100 at 75° C., i.e. both at conditions above the cloud point of the respective surfactant, Triton X-100 results in better LPS removal. At the same temperature (at 57.5° C., i.e. below the cloud point of Triton X-100 but above that of Triton X-114) the difference in LPS removal is even more pronounced.

[0125] From a mixing experiment, it is clear that the more relative amount of Triton X-100 as compared to the of Triton X-114, the better the LPS removal is.

TABLE-US-00010 TABLE 7 Triton X114 an Triton X100 as surfactant Amount Triton Amount Triton LPS Temperature X100 (%)* X114 (%)* (EU/g) (° C.) 100 0 26.8 >75 100 0 15.0 <60 80 20 19.8 <60 50 50 44.5 <60 20 80 85.0 <60 0 100 150-114 <60 *Mix ratios of Triton X100 and Triton X114 to obtain a total of 0.18% w/w surfactant solution. ** 75° C. is above and <60° C. (i.e. 57.5° C.) is below the Triton X100 cloud point.

Example 8

[0126] Effect of Gelatin Concentration on LPS Removal.

[0127] A test as described in example 1 has been performed with gelatin 7 wherein the gelatin concentration varied from 6.66 w/w % to 10 w/w % and 15 w/w %, see table 8. The Triton X100 concentration was increased in equal ratio to the gelatin concentration. For a 6.67% gelatin concentration 1.4 g (0.18 w/w %) Triton X100 was applied. For a 10 w/w % gelatin solution 2.1 g (0.27 w/w %) and for a 15 w/w % gelatin solution 3.2 g (0.40 w/w %) Triton X100 was used. In line with Triton X100, also the active carbon amounts added were increased from 5 gram (0.7 w/w %) for the 6.66% gelatin solution to 7.5 gram (1.05 w/w %) and 11.3 gram (1.6 w/w %) for the 10 and 15% (w/w) gelatin solutions, respectively. Mixing during the various purification steps was done at 500-1000 rpm for 15-30 minutes. The gelatins were filtered using filtration over a 2 μm filter (Whatman® Glass microfiber filters GF/C grade, 55 mm diameter, 2 μm (Schleicher & Schuell), using a Buchner funnel. Gelatins were freeze-dried before LPS analysis as described before.

[0128] At higher gelatin concentrations, the filtration step to remove the activated carbon requires more efforts. To confirm possible remaining Triton X100 traces which can influence the LPS/LAL analysis, surface tension measurements were performed as described in example 4 above. The surface tension results are equal/close to the original gelatin containing no Triton X100.

TABLE-US-00011 TABLE 8 Effect of gelatin concentration on LPS removal, gelatin 7, pH 4.7 and 57.5° C. Gelatin Purified LPS Surface tension concentration (%) Level (EU/g) (mN/m) 6.66 15 65.7 10 16 65.0 15 50 65.0 Gelatin non treated 2950 66.1

[0129] The LPS purification is hardly affected by gelatin concentration. Also at high, 15 w/w % gelatin solutions an effective purification was measured.

Example 9

[0130] Comparison of Different Triton.

[0131] 6.66 w/w % gelatin solutions were prepared as described in example 1, using gelatin batch 5. 0.18 w/w % of different Triton species were added to the gelatin solutions followed by mixing at 55° C. for 30 minutes. Subsequently, 5.0 g (0.7 w/w %) active carbon (Norit SX-Plus) was added, mixed at 55° C. for 30 minutes at 500-1000 rpm and removed as described in example 1, using a 0.45 μm filter (Phenex RC 26 mm, 0.45 μm). After purification the gelatins were stored at −20° C. and freeze-dried as described in example 1. Purified and non-purified liquid gelatin samples were stored at −20° C. and freeze-dried before LPS analysis.

[0132] It was observed that with Triton species having the formula C.sub.8H.sub.15—C.sub.6H.sub.4—O—(C.sub.2H.sub.4O).sub.nH wherein n is between 8 and 13, a low LPS content of 20 EU or less could be achieved. The purified gelatin had a molecular weight and a viscosity comparable to the initial gelatin before purification.

TABLE-US-00012 TABLE 9 LPS reduction with different Triton at 55° C. Triton LPS level (EU/g) N number Triton X165 350 16 Triton X102 15 12 Triton X100 10 9.5 Triton X114 150 7.5 Non-treated gelatin 5 5200

[0133] FIG. 3 shows a graph wherein Triton X100, Triton X102, Triton X114 and Triton X165 are used in the purification of gelatin. The graph shows that when the n value lies between 8 and 13, specifically between 8.5 and 12.5 a better purification is obtained of about 20 EU/g or less. It is noted that the use of both Triton X-114 (n of 7.5) and Triton-X165 (n of 16) also resulted in reducing the level of the LPS content in a gelatin-containing LPS but not to the levels obtained with Tritons having an “n” value between 8 and 13 such as Triton X-100 and Triton X-102.

Example 10

[0134] Purification of Type B Bone Gelatin.

[0135] The test conditions are equal to the previous described examples.

[0136] A 6.66% (w/w) gelatin 12, 13 and 14 solution was prepared and the temperature was kept at 57.5° C. pH of the gelatin solution was not adjusted. Triton X100 was added to a concentration of 0.18 w/w % and mixed for 30 minutes. Subsequently, an amount of 5.0 g (0.7 w/w %) active carbon was added followed by an additional 15 minutes mixing. Finally the gelatin solutions was filtered as described before. Temperature was kept at 57.5° C. The LPS level of the filtered gelatin solution was measured directly or first frozen at −20° C. followed by freeze-drying as described in the previous examples.

[0137] The Triton X100 purification method is also very effective to purify type B gelatins to levels below 20 EU/g. Lower purified LPS levels are obtained in case the LPS level in the starting gelatin is lower.

TABLE-US-00013 TABLE 10 Purification of Type B gelatin Initial LPS Purified LPS gelatin pH level (EU/g) level (EU/g) 12 5.7 3200 70 13 5.8 6720 20 14 5.4 250 8.4

Example 11

[0138] Purification of Type B Bone Gelatin at Different pH Values.

[0139] The conditions were equal to the conditions used in example 10. Gelatin 12 solution were prepared and pH was adjusted to values between 4 and 6 using either 0.1M hydro-chloric acid or 0.1M sodium hydroxide, both from Sigma-Aldrich. Gelatins were freeze-dried before LPS analysis as described above.

TABLE-US-00014 TABLE 11 Type B, gelatin 15, purification at different pH values at 57.5° C. Gelatin (12) LPS content (EU/g) Non purified, initial LPS value 3200 Purified at pH 4.0 44 Purified at pH 4.5 44 Purified at pH 5.0 44 Purified at pH 6.0 71 Purified at pH 7.0 78

[0140] A pH effect is observed. Particular at values below 6.0, improved LPS purification was observed.

Example 12

[0141] Purification of Type a Fish Gelatin.

[0142] The test conditions were as described for examples 10 and 11.

[0143] A 6.66% (w/w) gelatin 11 solution was prepared at 57.5° C. and Triton X100 was added to a concentration of 0.18 w/w %. Subsequently, an amount of 5.0 g active carbon was added followed by an additional 15 minutes mixing. Finally the solution was filtered as described before. Temperature was kept at 57.5° C. The pH of the gelatin solution was not adjusted and was kept at 5.7. Gelatins were freeze-dried before LPS analysis as described before.

TABLE-US-00015 TABLE 12 Type A fish gelatin purification: Gelatin (11) LPS content (EU/g) Non purified 33760 LPS content after purification 80

[0144] The Triton X100 purification is very efficient to purify high LPS containing fish gelatin. It was observed that the level of purification for the type A fish gelatin can further be improved by decreasing the pH of the fish gelatin solution to values between 4.5 and 5.5. An additional purification by repetition of steps 2)-5) can be executed to obtain levels <20/<10/<5 EU/g, as result of the initial high LPS level.

Example 13

[0145] Different Active Carbon Removal Methods

[0146] A gelatin 7 solution was prepared as mentioned in the previous examples.

[0147] A Triton X100 concentration 0.18 w/w % was applied. The solution was mixed for 30 minutes at 750 rpm at 57.5° C. Next, 5 gram (0.7 w/w %) active carbon was added followed by an additional 15-30 minutes mixing at 57.5° C. After incubation the active carbon was removed in three different ways:

[0148] 1. Filtration over a 0.45 μm filter, as described in previous examples

[0149] 2. Filtration over a 2 μm filter (Whatman® Glass microfiber filters GF/C grade, 55 mm diameter, 2 μm (Schleicher & Schuell), using a Buchner funnel. Larger filter pores are beneficial for processing.

[0150] 3. Filtration over non activated diatomaceous earth (Clarcel CBL, Ceca Chemicals, France, or Sigma-Aldrich D3877, Sigma-Aldrich, USA), using a Buchner funnel. 7.5-10 gram diatomaceous earth was used per 125 gram 6.67% gelatin solution containing 0.18 w/w % Triton X100 and 0.7 w/w % active carbon. Diatomaceous earth is a well-known filtration aid, e.g. used in the gelatin production process.

[0151] Gelatins were freeze-dried before LPS analysis as described before.

[0152] The surface tension of the filtered gelatine solutions mentioned in table 13 was 66-67 mN/m, equal to the non-treated gelatin control solution (data not shown).

[0153] All 3 filtration methods can be used to effectively remove active carbon and the adsorbed Triton X100 and LPS. The results suggest that filtration over diatomaceous earth is more effective compared to a 0.45 μm and a 2 μm filter and suggests an additional LPS purification by diatomaceous earth.

TABLE-US-00016 TABLE 13 Different active carbon removal methods Gelatin (7) LPS content (EU/g) Non-treated gelatin 2850 Purified, active carbon filtration 34 according to method 1 Purified, active carbon filtration 17 according to method 2 Purified, active carbon filtration 1.5 with diatomaceous earth

[0154] In another experiment, the active carbon was not introduced into the gelatin solution, but the aqueous medium comprising both gelatin and the surfactant were passed through a 3M ZetaCarbon filter cartridge of the R55S type (3M, USA), resulting in improved LPS removal as compared to the above method 2. Also, the surface tension of the solution after passing the filter was 66-67 mN/m, equal to the non-treated gelatin control solution, indicating that the surfactant substantially completely remained in the filter, see also example 4. In cases where the surface tension is not similar to the control solution, two or if desired more filter cartridges can be used in series. Similar results were found when instead of the R55S filter, a filter of the R30L3S type (3M, USA) was used.

Example 14

[0155] Effects of Triton X100, Oxidation and Combined Triton X100-Oxidation

[0156] Three different 6.66% gelatin 1 and gelatin 10 solutions were prepared according the methods described in example 1. To one solution Triton X100 was added to a concentration of 0.18 w/w %. To the second solution, H.sub.2O.sub.2 was added to a concentration of 1.5 w/w %. To the third solution, Triton X100 and H.sub.2O.sub.2 were added to the concentration of 0.18 w/w % and 1.5% w/w % respectively. pH of the solutions were not adjusted and were 5.5 and 5.3 for gelatin 1 and gelatin 10 respectively. Mixing and incubation was done at 57.5° C. for 30 minutes. A minimum of 5 gram (0.7 w/w %) active carbon was added to gelatin solutions followed by an additional 15-30 minutes mixing. Subsequently, the gelatin solutions were filtered using a 0.45 μm filter (Phenex RC 26 mm, 0.45 μm) as described in example 1. Gelatins were freeze-dried before LPS analysis as described above. Remaining H.sub.2O.sub.2 was analyzed using the method described in the GME10 and was <20 ppm, confirming that no interference with the LAL analysis method occurred.

TABLE-US-00017 TABLE 14 Oxidizing agents - Triton X100 effect with gelatin 1 and gelatin 10 Treatment LPS content (EU/g) Gelatin 1 Initial 1075 Triton X100 2 1.5% H.sub.2O.sub.2 122 Triton X100 + H.sub.2O.sub.2 <1 Gelatin 10 Initial 17640 Triton X100 288 1.5% H.sub.2O.sub.2 3116 Triton X100 + H.sub.2O.sub.2 18

[0157] From Hirayama and Sakata, supra, U.S. Pat. No. 8,133,269 and WO2012031916 it is known that H.sub.2O.sub.2 reduces or inactivates LPS in gelatin. However, we now observe a surprising synergetic Triton X100 and H.sub.2O.sub.2 effect.

Example 15

[0158] Gelatin and Triton X100 Variation and Effect on LPS Purification

[0159] Gelatin solutions with different concentions of dissolved gelatin were mixed at different concentrations of Triton X-100 under constant mixing with a speed of 750 rpm and at a temperature of 55 to 57.5° C. for 30 minutes. The mixture was then filtered over two 3M ZetaCarbon filter cartridges, of type R55s. The filtrate is collected for direct LPS analysis, or frozen at −20° C. and freeze dried using a Christ Alpha 2-4LD Plus freeze-dryer (MartinChrist, Germany). Freeze-drying vacuum conditions: 0.04 mbar and −87° C. for at least 24-48 hours until the solutions are dried to a moisture content of around 4-6%. No moisture correction was done before endotoxin analysis.

TABLE-US-00018 TABLE 15 Gelatin and Triton X100 variation and effect on LPS purification Initial LPS Gelatin Triton X100 Purified LPS Sample level (EU/g) w/w % w/w % level (EU/g) 1 1149 13 0.3 5 2 4439 13.8 0.32 29 3 4179 15.5 0.38 32 4 4921 12.9 0.32 15 5 10074 20.4 0.4 10 6 9565 22 0.5 13 7 11467 20.4 0.4 8 8 10376 21.6 0.4 26