METHODS FOR THE PRODUCTION OF BIOPOLYMERS WITH SPECIFIC MOLECULAR WEIGHT DISTRIBUTION

Abstract

The present invention relates to a method for the production of a biopolymer, wherein the biopolymer has a defined average molecular weight, the method comprising lyophylizing a composition comprising the biopolymer with native high molecular weight, optionally purifying and/or isolating the biopolymer; wherein the temperature during the sublimation process is selected to facilitate a controlled and defined degradation of said biopolymer.

Claims

1. A method for the production of a biopolymer composition comprising at least one biopolymer, wherein the at least one biopolymer has a defined average molecular weight, the method comprising: providing a first composition comprising a biopolymer with a defined pH-value; (ii) providing a second composition comprising a biopolymer with a defined pH-value; (iii) combining the first composition and the second composition in a reaction vessel without substantially mixing the first and second compositions with each other; (iv) optionally freezing the first and second compositions; (v) lyophylizing the compositions comprising the at least one biopolymer, (v) optionally purifying and/or isolating the at least one biopolymer; wherein the maximum temperature during the lyophilization process is selected to facilitate a controlled and defined degradation of said biopolymer and wherein water is removed by sublimation; wherein the pH-values of the first and second composition differ by at least 0.1.

2. The method according to claim 1, wherein the first and second composition comprise the same biopolymer.

3. The method according to claim 1, wherein the first and second composition comprise different biopolymers.

4. The method according to claim 1, wherein the first or second composition comprising a biopolymer is a frozen aqueous solution or an emulsion.

5. The method according to claim 1, wherein the pH value of the first or second composition is between 1.5 and 8.5.

6. The method according to claim 1, wherein the temperature during the sublimation process is between 40 C. and 150 C.

7. The method according to claim 1, wherein the pressure during the sublimation process is between 50 bar and 800 bar.

8. The method according to claim 1, wherein the biopolymer of the first or second composition is hyaluronic acid, collagen, a glucosamino glycan, a polysaccharide or a fucoidane.

9. The method according to claim 1, wherein the first or second composition is an emulsion comprising: (i) a biopolymer, (ii) water, (iii) optionally a pharmaceutically, dermatologically and/or cosmetically acceptable compound or oil, (iv) optionally an emulsifying agent and (v) optionally emollients.

10. The method according to claim 1, wherein the first or second composition further comprises dermatological, pharmaceutical or cosmetic additions.

11. The method according to claim 1, wherein the first or second composition comprises additional components, so that the resulting lyophilized product is easily dissolvable or easily forms an emulsion.

12. The method according to claim 1, wherein the first and second composition comprise the same contents and differ in pH-value or and/or the biopolymer.

13. The method according to claim 1, wherein the first and second composition are combined in an appropriate container, which is suitable for the lyophilization process and which can serve as primary packaging.

14. The method according to claim 1, wherein the biopolymer of the first and second composition is hyaluronic acid, collagen, a glucosamino glycan, a polysaccharide and a fucoidane.

15. The method according to claim 1, wherein the biopolymer composition is a pharmaceutically, dermatologically or cosmetically acceptable biopolymer composition.

16. A composition produced according to claim 1.

17. The composition according to claim 16, wherein the composition is a pharmaceutical, dermatological or cosmetic product.

18. (canceled)

Description

FIGURE LEGENDS

[0092] FIG. 1: Correlation of average molecular weight, skin penetration and biological activity of hyaluronic acid.

[0093] FIG. 2: Correlation of pH, temperature and obtained average molecular weight obtained, when processing a composition comprising hyaluronic acid according to the present invention.

[0094] FIG. 3: Overview of suggested temperature profiles over time during the lyophilization process.

[0095] FIG. 4: Calibration curve to determine average molecular weight of hyaluronic acid, processed according to the present invention.

[0096] FIG. 5: Elution and molecular mass profiles of neutral oil (a) and Sepinov EMT-10 (b) processed according to the present invention.

[0097] FIG. 6: comparison of the elution profiles (A) and molecular mass profiles (B) of native hyaluronic acid and hyaluronic acid, lyophilized 120 C.

[0098] FIG. 7: Comparison of the elution profile (A) and molecular mass profiles (B) of a mixture of hyaluronic acid, Sepinov EMT-10 and neutral oil, lyophilized at different temperatures.

[0099] FIG. 8: Molecular weight of lyophilized samples of 1 wt-% high molecular weight HA samples with pH adjusted in the range of 6.21 to 2.9.

[0100] FIG. 9: Molecular weight distributions of lyophilized samples of 1 wt-% high molecular weight HA samples with pH adjusted to 6.21 and 2.9.

[0101] FIG. 10: Molecular weight of hyaluronic acid containing emulsions lyophilized at different process temperatures.

[0102] FIG. 11: Molecular weight distributions of lyophilized samples of 1 wt-% high molecular weight HA samples with pH adjusted to 6.21 to 2.9 stacked at different volume ratios

[0103] FIG. 12: Molecular weight distributions of lyophilized samples of 1 wt-% pullulan samples with pH adjusted to 4.9 to 3.5 stacked at different volume ratios

[0104] FIG. 13: Molecular weight distributions of lyophilized samples of 1 wt-% sodium alginate samples with pH adjusted to 3.5 and 7.15 stacked at different volume ratios

EXAMPLES

Example 1: Controlled Degradation of Hyaluronic Acid

[0105] Deionized water is transferred to 1 l lab reactor and stirred at 75 C. Hyaluronic acid powder is added and stirred at 75 C. at 700 rpm for 15 min until the material is dissolved. The emulsifier component is added and stirred at 50 C. for 15 min at 1400 rpm under reduced pressure (200 bar). The oil component is added and stirred at 1400 rpm/45 C./200 bar for 10 min and subsequently for 5 min at 2100 rpm/45 C./200 bar. The received emulsion is cooled to room temperature and transferred to 10 ml glass vials and stored overnight at ambient conditions. Samples were frozen in a deep freezer for minimum 16 h and subsequently lyophilized up to maximum target temperature.

[0106] As a proof of principle hyaluronic acid was processed according to the invented method. Herein, pure hyaluronic acid, and compositions of hyaluronic acid with MCT neutral oil and Sepinov EMT-10 were lyophilized at varying temperatures.

[0107] The following samples were analyzed: [0108] 1. Neutral oil, unprocessed [0109] 2. Sepinov EMT-10, unprocessed [0110] 3. Hyaluronic acid, unprocessed [0111] 4. Sepinov EMT-10, lyophilized at 120 C. [0112] 5. Hyaluronic acid, lyophilized at 120 C. [0113] 6. Mixture (hyaluronic acid, neutral oil and Sepinov EMT-10), lyophilized at 40 C. [0114] 7. Mixture, lyophilized at 60 C. [0115] 8. Mixture, lyophilized at 80 C. [0116] 9. Mixture, lyophilized at 100 C. [0117] 10. Mixture, lyophilized at 120 C.

[0118] The samples were analyzed using size exclusion chromatography on an HPLC system, using 3 analytical columns. Samples were dissolved in PBS-Buffer with pH 7.4, non-soluble parts were removed by filtration.

[0119] The columns were calibrated using dextran/pullulan standards. Molecular masses of the samples were determined based on the said calibration (for the calibration curve see FIG. 4).

[0120] Only pure hyaluronic acid samples were completely soluble. The soluble components of the Sepinov EMT-10 or neutral oil, do not produce any problematic signals during analysis (see FIG. 5 a and b).

[0121] The results clearly show that the composition and the lyophilization temperature affect the average molecular weight of the hyaluronic acid. While an effect of lyophilization on pure hyaluronic acid at high temperatures occurs and results in a reduced average molecular weight (see FIGS. 6 a,b), the effect is stronger in the mixtures (see FIG. 7 a,b).

[0122] Overall it is clearly visible that the choice of parameters during the lyophilization process is suitable to control the average molecular weight of hyaluronic acid after the lyophilization.

Example 2: Influence of the pH-Value on Degradation

[0123] Hyaluronic acid with a molecular weight of 1.478 Mio Da (Contipro, Mw, according to gel permeation chromatography) was dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. The pH was adjusted with hydrochloric acid in the range of 2.9 to 6.21.

[0124] 7.5 ml HA solution was dispensed in 10 ml glass vials, samples were frozen at 20 C. overnight and placed in a Christ Epsilon 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. temperature profile shown in FIG. 3.

[0125] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analyzed by means of gel permeation chromatography against Pullulan and Dextran molecular weight standards.

[0126] Independent on adjusted pH, all samples were cleaved showing a maximum of 766 kDa at pH 6.21 and a minimum 84.75 kDa at pH 2.9 (FIG. 7). The higher the amount of free acid functionality in the polymer the higher the tendency of the polymer to be cleaved. A corresponding elugram of the high as well as the low molecular weight sample is shown in FIG. 8.

Example 3: Degradation of Hyaluronic Acids with Different Molecular Weights

[0127] Four differents types of hyaluronic acid (Contipro/GfN 3010 (MW: 1478 kDa), Principium Cube3 (MW: 733 kDa), Principium Signal-10 (MW: 25 kDa) and Freda mini-HA (MW: 27 kDa)) were dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. Solution were used as is or pH was adjusted to approximately 3.5.

[0128] 7.5 ml HA solution was dispensed in 10 ml glass vials, samples were frozen at 20 C. overnight and placed in a Christ Epsilon 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. or alternatively the 120 C. temperature profile shown in FIG. 3.

[0129] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of gel permeation chromatography against Pullulan and Dextran molecular weight standards.

[0130] High and medium molecular weight hyaluronic acid showed a moderate decay of molecular weight at original pH dissolved in distilled water, whereas molecular weight of substances decayed drastically at low pH, as shown in the following table.

TABLE-US-00001 Mw [kDa] non- Mw [kDa]* processed at Mw [kDa]* processed processed pH 10/120 C. at 120 C. 1478 6.11 594 531 1478 3.50 97 68 733 6.63 331 297 733 3.57 97 78 25 3.36 23 21 27 6.53 nd 29 27 3.50 nd 28

Example 4: Emulsions Containing Hyaluronic Acid

[0131] 5 g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in 465 g of distilled water, heated to 80 C. and stirred by means of a Somakon MP-LB (11) mixing device at 1400 rpm and ambient pressure for 15 minutes.

[0132] 7.5 g Sepinov EMT-10 (INCI name: Hydroxyethyl acrylate (and) Sodium Acryloyl Dimethyl Taurate Copolymer) was added the pH was adjusted to 3.05 and mixture was stirred at 1400 rpm/200 bar for further 15 minutes at 80 C.

[0133] 25 g of medium chain triglyciderides (MCTs) as model oil compound were added and homogenized at 2100 rpm/200 bar for 5 minutes.

[0134] 7.5 ml of the resulting emulsion was dispensed in 10 ml glass vials, samples were frozen at 20 C. overnight and placed in a Christ Epsilon 2-10D LSC plus HT device and processed for approximately 20 hours at maximum 40, 60, 80, 100 and 120 C.

[0135] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analyzed by means of GPC. FIG. 9 shows the temperature dependence of the molecular weight (Mw) of the hyaluronic acid decreasing with increasing maximum process temperature.

Example 5: Lyophilization of Different Biopolymers

[0136] Polymers were dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. The pH of the solutions was measured and the molecular weight distribution of the non-processed polymer solutions were determined by means of size exclusion chromatography against Pullulan and Dextran molecular weight standards diluting the samples to 0.3 wt-% in PBS buffer (pH 7.4).

[0137] 7.5 ml polymer solution was dispensed in 10 ml glass vials, samples were frozen at 20 C. overnight and placed in a Christ Epsilon 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. temperature profile shown in FIG. 3.

[0138] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of GPC. The results are shown in the following tables.

Sodium Alginates:

[0139]

TABLE-US-00002 Mw [kDa] Mw [kDa] Mw [kDa]* non- processed at processed at processed pH 10/120 C. 120 C. 1074 6.88 336 269 1074 3.50 239 nd 1020 7.03 472 336 1020 3.50 287 nd 881 7.15 259 233 881 3.50 184 nd

Polysaccharides:

[0140]

TABLE-US-00003 Mw [kDa]** Mw [kDa]* processed Mw [kDa]** non- at processed at Polymer Monomers pH processed 10/120 C. 120 C. Rhizobian Gum tbd 5.59 706 533 592 3.50 706 354 nd Sodium carboxy Funktionalized 6.66 682 689 712 methyl cellulose glucose 3.5 682 309 308 Pullulan Glucose 5.46 314 239 278 (Maltotriose) 3.50 314 61 nd Biosaccharide Fucose 7.35 2037**** 1735**** 1598**** Gum-1 3.50 2037**** 443 nd Glucomannane Glucose, mannose 5.84 1304 1119 980 3.50 1304 247 nd Beta-Glucan Galacturonic acid, 4.02 778 389 358 (and) Pectin rhamnose Tamarindus Glucose, xylose, 6.20 956 933 927 indica Seed galoctoxylose Polysaccharide 3.50 956 602 nd

Example 6: pH and Volume Variation with Stacked Hyaluronic Acid Solutions

[0141] Hyaluronic acid with a molecular weight of 1.478 Mio Da (Contipro, Mw, according to gel permeation chromatography) was dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. One fraction of the solution was used at normal pH, the second fraction was adjusted with hydrochloric acid to pH 2.9.

[0142] pH 6.21 HA solution was dispensed in differed volumes from 0.75 to 6.75 ml in 10 ml glass vials. Samples were frozen at 20 C. and stacked with pH 2.9 HA solution at 6.75 to 0.75 ml and frozen again and stored at 20 C. overnight. The corresponding volume ratios are shown in the following table:

TABLE-US-00004 Volume Volume Weight-% Weight-% pH 6.21 pH 2.9 pH 6.21 pH 2.9 Mw [kDa] of solution [ml] solution [ml] solution solution mixture 6.75 0.75 90 10 595 5.625 1.875 75 25 515 3.75 3.75 50 50 417 1.875 5.625 25 75 201 0.75 6.75 10 90 184

[0143] Samples were placed in a Christ 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. temperature profile shown in FIG. 3.

[0144] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of gel permeation chromatography against Pullulan and Dextran molecular weight standards.

[0145] Dependant on volume ratio differently shaped molecular weight distributions can be shaped (see FIG. 10), indicating that dependent on the volume fractions and the adjusted pH in the volume fractions any shape of molecular weight distribution can be achieved. Dependant on targeted biological function an optimum molecular weight distribution can be designed.

Example 7: pH and Volume Variation with Stacked Pullulan Solutions

[0146] Pullulan with a molecular weight of 371 kDa (Hayashibara, Mw, according to gel permeation chromatography) was dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. One fraction of the solution was used at normal pH (4.9), the second fraction was adjusted with hydrochloric acid to pH 3.5.

[0147] pH 4.9 pullulan solution was dispensed in differed volumes from 0.75 to 6.75 ml in 10 ml glass vials. Samples were frozen at 20 C. and stacked with pH 2.9 pullulan solution at 6.75 to 0.75 ml and frozen again and stored at 20 C. overnight. The corresponding volume ratios are shown in the following table.

TABLE-US-00005 Volume Volume Weight-% Weight-% pH 4.9 pH 3.5 pH 4.9 pH 3.5 Mw [kDa] of solution [ml] solution [ml] solution solution mixture 6.75 0.75 90 10 176 5.625 1.875 75 25 133 3.75 3.75 50 50 94 1.875 5.625 25 75 62 0.75 6.75 10 90 36

[0148] Samples were placed in a Christ 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. temperature profile shown in FIG. 3.

[0149] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of gel permeation chromatography against Pullulan and Dextran molecular weight standards.

[0150] Dependant on volume ratio differently shaped molecular weight distributions can be achieved (see FIG. 11). Molecular weight Mw is mainly influenced by the amount of the low pH solution.

Example 8: pH and Volume Variation with Stacked Sodium Alginate Solutions

[0151] Sodium alginate with a molecular weight of 881 kDa (Cargill, Mw, according to gel permeation chromatography) was dissolved in 1 wt-% solution in distilled water at 80 C. for five minutes. One fraction of the solution was used at normal pH (7.15), the second fraction was adjusted with hydrochloric acid to pH 3.5.

[0152] pH 7.15 sodium alginate solution was dispensed in differed volumes from 0.75 to 6.75 ml in 10 ml glass vials. Samples were frozen at 20 C. and stacked with pH 3.5 sodium alginate solution at 6.75 to 0.75 ml and frozen again and stored at 20 C. overnight. The corresponding volume ratios are shown in the following table.

TABLE-US-00006 Volume Volume Weight-% Weight-% pH 7.15 pH 3.5 pH 7.15 pH 3.5 Mw [kDa] of solution [ml] solution [ml] solution solution mixture 6.75 0.75 90 10 357 5.625 1.875 75 25 315 3.75 3.75 50 50 278 1.875 5.625 25 75 253 0.75 6.75 10 90 245

[0153] Samples were placed in a Christ 2-10D LSC plus HT device and processed for approximately 20 hours according to the 10/120 C. temperature profile shown in FIG. 3.

[0154] Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of gel permeation chromatography against Pullulan and Dextran molecular weight standards. Dependent on volume ratio differently shaped molecular weight distributions can be shaped (see FIG. 12). Shift in molecular weight is mainly affected by the lyophilisation conditions and less by the amount of the low pH solution.