Methods for the production of biopolymers with defined average molecular weight

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 of making a biopolymer having a defined average molecular weight, the method comprising: providing a composition comprising a biopolymer, wherein the biopolymer is selected from the group consisting of hyaluronic acid having a molecular weight of 1.5-0.025 MDa, collagen, glucosamino glycans, polysaccharides and fucoidanes; and lyophilizing the composition comprising the biopolymer to remove water from the composition by sublimation, and to facilitate a controlled and defined degradation of the biopolymer to form the biopolymer having the defined average molecular weight; (i) wherein the lyophilization process for hyaluronic acid having a molecular weight of 1.5-0.025 MDa occurs over two temperatures, wherein the first temperature is 10° C. and the second temperature is 120° C.; and wherein the composition prior to lyophilization has a pH value between 6.63 and 3.36 to facilitate the controlled and defined degradation of the hyaluronic acid to hyaluronic acid having the defined average molecular weight in a range between 594 kDa and 23 kDa, (ii) wherein the lyophilization process for collagen, glucosamino glycans, polysaccharides, and fucoidanes occurs over two temperatures, wherein the first temperature is 10° C. and the second temperature is 120° C., and wherein the composition prior to lyophilization has a pH value between 1.5 and 8.5 to facilitate the controlled and defined degradation of collagen, glucosamino glycans, polysaccharides, and fucoidanes to collagen, glucosamino glycans, polysaccharides, and fucoidanes, respectively, having the defined average molecular weight; and (iii) wherein the method does not comprise purifying the biopolymer having the defined average molecular weight after lyophilization.

2. The method according to claim 1, wherein the composition comprising the biopolymer is an aqueous solution or an emulsion.

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

4. A method of making a composition, comprising hyaluronic acid having a defined average molecular weight, the method comprising: (i) providing a base composition comprising hyaluronic acid having a molecular weight of 1.5-0.025 MDa, wherein the base composition has a pH between 6.63 and 3.36; and (ii) lyophilizing said base composition over two temperatures, wherein the first temperature is 10° C. and the second temperature is 120° C., and wherein the method facilitates a controlled and defined degradation of hyaluronic acid to form hyaluronic acid having the defined average molecular weight in a range between 594 kDa and 23 kDa.

5. The method according to claim 4, wherein the base composition is an emulsion or an aqueous solution.

6. The method according to claim 5, wherein the base composition is an emulsion comprising: (i) hyaluronic acid having a molecular weight of 1.5-0.025 MDa, (ii) water, (iii) a pharmaceutically, dermatologically or cosmetically acceptable oil, (iv) an emulsifying agent and (v) one or more emollients.

7. The method according to claim 6, wherein the base composition further comprises dermatological, pharmaceutical or cosmetic ingredients.

8. The method according to claim 4, wherein the base composition comprises additional components, so that the resulting lyophilized product is dissolvable or emulsifiable.

9. The method according to claim 4, wherein the composition produced is a pharmaceutically, dermatologically or cosmetically acceptable composition.

Description

FIGURE LEGENDS

(1) FIG. 1: Correlation of average molecular weight, skin penetration and biological activity of hyaluronic acid.

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

(3) FIG. 3: Overview of suggested temperature profiles over time during the lyophilization process.

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

(5) FIG. 5: Elugram and molecular mass profiles of neutral oil (5A) and Sepinov EMT-10 (5B) processed according to the present invention.

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

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

(8) 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.

(9) 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.

(10) FIG. 10: Molecular weight of hyaluronic acid containing emulsions lyophilized at different process temperatures.

EXAMPLES

Example 1

Controlled Degradation of Hyaluronic Acid

(11) 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 μbar). The oil component is added and stirred at 1400 rpm/45° C./200 μbar for 10 min and subsequently for 5 min at 2100 rpm/45° C./200 μbar. 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.

(12) 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.

(13) The following samples were analyzed:

(14) 1. Neutral oil, unprocessed

(15) 2. Sepinov EMT-10, unprocessed

(16) 3. Hyaluronic acid, unprocessed

(17) 4. Sepinov EMT-10, lyophilized at 120° C.

(18) 5. Hyaluronic acid, lyophilized at 120° C.

(19) 6. Mixture (hyaluronic acid, neutral oil and Sepinov EMT-10), lyophilized at 40° C.

(20) 7. Mixture, lyophilized at 60° C.

(21) 8. Mixture, lyophilized at 80° C.

(22) 9. Mixture, lyophilized at 100° C.

(23) 10. Mixture, lyophilized at 120° C.

(24) 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.

(25) 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).

(26) 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 FIGS. 5a and b).

(27) 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 FIG. 6a,b), the effect is stronger in the mixtures (see FIG. 7a,b).

(28) 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

(29) 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.

(30) 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.

(31) 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.

(32) 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

(33) 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. Solutions were used as is or pH was adjusted to approximately 3.5.

(34) 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.

(35) 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.

(36) High and medium molecular weight HAs 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.

(37) 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

(38) 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 (1 l) mixing device at 1400 rpm and ambient pressure for 15 minutes.

(39) 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 μbar for further 15 minutes at 80° C.

(40) 25 g of medium chain triglyciderides (MCTs) as model oil compound were added and homogenized at 2100 rpm/200 μbar for 5 minutes.

(41) 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.

(42) 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

(43) 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).

(44) 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.

(45) 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.

(46) Sodium alginates:

(47) TABLE-US-00002 Mw [kDa] Mw [kDa]* non- Mw [kDa] 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

(48) Polysaccharides:

(49) 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

Lyophilisation of a Hyaluronic Acid Emulsions Containing Different Emulsifying Polymer Components

(50) 3 g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in 277.5 g of distilled water heated to 80° C. and stirred by means of a Somakon MP-LB (1 l) mixing device at 1400 rpm and ambient pressure for 15 minutes.

(51) 4.5 g emulsifying polymer was added and the pH was adjusted to approximately 3 and mixture was stirred at 1400 rpm/200 mbar for further 15 minutes at 80° C.

(52) 15 g of medium chain triglyciderides (MCTs) were added and homogenized at 2100 rpm/200 mbar for 5 minutes.

(53) 7.5 ml of the resulting emulsions 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 120° C.

(54) The following table shows the emulsifying polymer used, the pH values as well as the viscosity of the emulsion (measured with a hand-held HAAKE Viscotester 2 plus) prior to lyophilisation. All freeze-dried samples provided fast rehydration to opaque emulsions.

(55) TABLE-US-00004 Emulsifying Viscosity* Polymer Manufacturer INCI name pH [Pas] EMT-10 SEPPIC Hydroxyethyl acrylate (and) Sodium 3.05 15 Acryloyl Dimethyl Taurate Copolymer P-88 SEPPIC Hydroxyethyl acrylate (and) Sodium 3.06 14.2 Acryloyl Dimethyl Taurate Copolymer Bergamuls Berg& Beta-Glucan (and) Pectin 3.03 6.2 Schmidt

Example 7

Lyophilisation of a Hyaluronic Acid Emulsions Containing Different Oil Components

(56) 3 g of high molecular weight HA (GfN/Contipro 3010, 1.5 MDa), 277.5 g of distilled water, 4.5 g EMT-10 as well as 15 g of oil component (MCT (Cosnaderm), Marula Oil (Seatons), Jojobaoil (J. H. Müller GmbH) or Argan oil (Seatons)) were processed as described in example 6.

(57) 7.5 ml of the resulting emulsions 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 120° C. All freeze-dried samples provided fast rehydration resulting in opaque emulsions.

Example 8

Lyophilisation of a Hyaluronic Acid Emulsions Containing a UV Filter Blend

(58) 3 g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in 277.5 g of distilled water heated to 80° C. and stirred by means of a Somakon MP-LB (1 l) mixing device at 1400 rpm and ambient pressure for 15 minutes. 4.5 g Sepinov EMT-10 was added and mixture was stirred at 1400 rpm/200 mbar for further 15 minutes at 80° C.

(59) 6 g Eusolex 9010 (Avobenzone), 15 g Eusolex OCR (Octocrylene) and 7.5 g Eusolex OR (Ethylhexyl Salicylate) were dissolved in 15 g of medium chain triglyciderides (MCTs) and the UV filter mixture was added to the polymer solution and homogenized at 2100 rpm/200 mbar for 5 minutes.

(60) 7.5 ml of the resulting emulsions 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 at 10/120° C. as shown in FIG. 3. Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed by means of GPC. Mw of the lyophilisate was measured as 975 kDa.

Example 9

Lyophilisation of a Hyaluronic Acid Emulsions Containing Microcrystalline Silver

(61) 3 g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in 277.5 g of distilled water heated to 80° C. and stirred by means of a Somakon MP-LB (1 l) mixing device at 1400 rpm and ambient pressure for 15 minutes.

(62) 4.5 g Sepinov EMT-10 was added and mixture was stirred at 1400 rpm/200 mbar for further 15 minutes at 80° C. 700 mg of microcrystalline silver was dispersed in 15 g of medium chain triglyciderides (MCTs) and the mixture was added to the polymer solution and homogenized at 2100 rpm/200 mbar for 5 minutes.

(63) 7.5 ml of the resulting emulsions 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 at 10/120° C. as shown in FIG. 3. All freeze-dried samples provided fast rehydration resulting in a colorless gel.