1
FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. ' METHODS FOR THE PRODUCTION OF BIOPOLYMERSWITH DEFINED AVERAGE
MOLECULAR WEIGHT '
2.
1. (A) MEDSKIN SOLUTIONS DR. SUWELACK AG
(B) Germany
(C) Josef-Suwelack-Strasse 48727 Billerbeck, Germany
The following specification particularly describes the invention and the manner in which it is to be
performed.
5
2
Field of the Invention
The present invention is in the field of dermatology, pharmaceutics and cosmetics. In particular
the invention is in the field of the production of pharmaceutically, dermatologically or
cosmetically applicable substances and in the 5 use and application thereof.
Background
Biopolymers, such as collagen, polysaccharides or hyaluronic acid are commonly used in cosmetic
10 or dermatological compositions. In many cases these biopolymers are used as moisturizers or
anti-oxidants. Common forms of administration are as cream, serum, patches, masks, balms,
liquids or as an ointment.
Hyaluronic acid or hyaluronan for example is a biopolymer, which is widely distributed among the
15 human tissue. It is an anionic, non-sulfated glycosaminoglycan comprising the following structure:
Hyaluronic acid has several medical uses, in particular in dermatology, and is commonly used in
20 cosmetic products, in particular so called anti-ageing products.
In general the bioactivity of biopolymers, such as hyaluronic acid is directly dependent on the
average molecular weight of said biopolymers. Taking hyaluronic acid and its use in dermatology
for example, the average molecular weight determines the depth of skin penetration and the
25 potential dermatological effects of hyaluronic acid (see Figure 1).
It is known, that the biological functionality of biopolymers is dependent on their average
molecular weight, several methods have been developed to generate biopolymers with defined
average molecular weight.
3
EP 2 479 194 A2 describes the hydrolysis of hyaluronic acid on activated charcoal. EP 2 463 309 B1
and EP 1 992 645 A1 describe several methods for the acidic hydrolysation of hyaluronic acid.
Other methods involve the use of enzymatic hydrolysis and filtration (EP 0 138 572 B1) or the use
of high temperatures and strong shearing 5 forces (EP 1 987 153 B1).
The problem with all these methods is, that in particular hyaluronic acid needs extensive
purification steps to remove the low molecular weight hyaluronic acids, which can be proinflammatory.
10
It is therefore necessary to provide a method for the efficient production of pure biopolymers
with defined molecular weight distribution, in particular hyaluronic acid, which allows the control
of the average molecular weight of the biopolymer and does not need any further additional
purification steps.
15
Brief Description of the Invention
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
20 (i) lyophilizing a composition comprising the biopolymer,
(ii) optionally purifying and/or isolating the biopolymer, wherein the temperature during the
lyophilization process is selected to facilitate a controlled and defined degradation of said
biopolymer.
25 In a preferred embodiment the invention relates to a method for the production of a biopolymer,
wherein the biopolymer has a defined average molecular weight, the method comprising
(i) providing a composition comprising a biopolymer, wherein optionally the pH of the
composition has been adjusted to between pH 1.5 and 8.5;
(ii) lyophilizing a composition comprising the biopolymer with native high molecular weight,
30 wherein the lypholization temperature is between -40 °C and 150 °C
(iii) optionally purifying and/or isolating the biopolymer,
wherein the temperature during the lyophilization process is selected to facilitate a controlled
and defined degradation of said biopolymer.
4
The invention further relates to the use of said method for the production of biopolymers and to
biopolymers, which are produced by said method.
In another embodiment the invention relates to a method for the production of compositions,
comprising a biopolymer, wherein the biopolymer has a defined average 5 molecular weight, the
method comprising:
(i) providing a base composition comprising a biopolymer,
(ii) lyophilizing said composition;
wherein the temperature during the sublimation process is selected to facilitate a controlled and
10 defined degradation of the biopolymer.
The invention further relates to the use of said method for the production of compositions
comprising a biopolymer and to compositions produced by said method.
15 Definitions
In the context of the present invention, biopolymers are polymers produced by living organisms.
The present invention only relates to native high molecular weight biopolymers, which are
preferably not technically or chemically modified, besides the common and native modifications,
20 which occur in the living organism. As polymers, they are characterized by repetitive monomeric
motives.
In general biopolymers are divided into three main classes: polynucleotides, polypeptides and
polysaccharides. Within the context of this invention the term “biopolymer” only refers to
25 polypeptides and polysaccharides. In the present invention the term “biopolymer” encompasses
all naturally occurring modifications of biopolymers, e.g. glycosylation, partial hydrolysis or the
attachment of lipids to polypeptides.
Polymers consisting of biological units, but not produced in a living organism, such as polylactic
30 acid, are not considered biopolymers within the meaning of the invention. Biopolymers according
to the above mentioned definition processed according to the present invention, are biopolymers
in the context of the present invention.
5
Non-limiting examples for biopolymers according to the present invention comprise: collagens,
starch, cellulose derivatives, glucosamino glycans, polysaccarides or fucoidanes.
In the context of the present invention lyophilization or lyophilizing refers to a dehydration
process, wherein water is removed by sublimation. Lyophilization 5 is commonly referred to as
freeze drying. In general lyophilization comprises three stages:
(i) freezing the composition to be dehydrated, wherein it is important that the composition
is cooled below its triple point. The suitable freezing method is dependent on the
10 components of the composition.
(ii) a primary drying phase, in which most of the water is removed, wherein the pressure is
lowered to a few μbar or even lower. In this stage the temperature is usually adjusted to
get the water sublimated. Preferably, but not necessarily, at this stage the temperature
remains under 0°C.
15 (iii) a secondary drying phase, wherein the pressure is optionally reduced, down to the range
of μbar and the temperature preferably raised above 0 °C to remove more strongly bound
water.
In one embodiment of the invention, the temperature is controlled during the second drying
20 phase. In another embodiment the temperature is controlled during the primary drying phase. In
a particular embodiment the composition is dried using only one drying step, wherein the
conditions correspond to the conditions of the second drying step. In an alternative embodiment
the composition is dried using only one drying step, wherein the conditions correspond to the
conditions of the first drying step.
25
Within the meaning of the present invention the “temperature during the sublimation process”
refers to the temperature of the storage plate on which the composition is placed.
In the context of the present invention the term aqueous solution refers to a solution, wherein
30 the solvent is water. Within the context of the present invention the term further refers to coarse
or colloidal suspensions of components, for example non-water-soluble biopolymers or nonsoluble
cosmetic additions in water.
6
In the context of the present invention the term emulsion refers to mixtures of normally
immiscible liquids. In the context of the present invention the term emulsion in particular refers
to water-in-oil or oil-in-water emulsion. Preferably in the context of the present invention the
emulsion is stabilized by the use of an emulsifying agent or emulsifier. Non-limited examples for
emulsifying agents are lecithin, sodium stearoyl lactylate, polymers 5 with emulsifying
functionalities or detergents.
Detailed description of the Invention
10 The inventors found surprisingly, that biopolymers can be subjected to controlled degradation
during lyophilization processes, resulting in biopolymers with defined average molecular weight.
A first aspect of 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
15
(i) lyophilizing a composition comprising the biopolymer,
(ii) optionally purifying and/or isolating the biopolymer, wherein the maximum temperature
during the lyophilization process is selected to facilitate a controlled and defined
degradation of said biopolymer.
20
The composition comprising the biopolymer can be any kind of composition, provided said
composition comprises at least small amounts of water in addition to said biopolymer. Said
composition may comprise additional biopolymers, i.e. mixtures.
25 The method is in particular suitable for biopolymers selected from the group comprising
hyaluronic acid, collagen, glucosamino glycans, polysaccharides and fucoidanes. In a preferred
embodiment the biopolymer is a glucosamino glycan or polysaccarid. In a more preferred
embodiment the biopolymer is selected from the group consisting of alginates, rhizobian gum,
sodium carboxy methyl cellulose, pullulan, Biosaccharide Gum-1, glucomannane, beta-glucane,
30 pectine, tamarindus indica seed polysaccharide and hyaluronic acid. In an even more preferred
embodiment the biopolymer is sodium alginate or hyaluronic acid. In the most preferred
embodiment the biopolymer is hyaluronic acid.
7
In a preferred embodiment the biopolymer is a biopolymer with high molecular weight. In a more
preferred embodiment the biopolymer is a biopolymer with native high molecular weight.
In a preferred embodiment the composition comprising the biopolymer is an aqueous solution or
5 an emulsion.
In another embodiment of the present invention the composition comprising the biopolymer is a
gel or a liquid with low to high viscosity.
10 The inventors had found in particular, that controlled conditions during the sublimation process
and a control of the parameters of the composition, e.g. salt contents, pH-value, vacuum, used
emulsifying agents, allow the control of the average molecular weight of the degraded
biopolymer.
15 In one embodiment of the invention the composition comprising the biopolymer has a pH-value
selected from a range between 1.5 and 8.5. In a preferred embodiment the pH value is selected
from a range between 2.5 and 6.
The inventors had found a direct correlation between the pH-value, the temperature during the
20 sublimation process and the average molecular weight of the biopolymer. Figure 2 shows the
correlation found for an analyzed hyaluronic acid.
It is evident that the average molecular weight of the final product of the processed biopolymer is
directly dependent on the combination of temperature and pH value selected.
25
In one embodiment of the invention the maximum temperature during the sublimation process is
selected from the range of – 40 °C to 150 °C. In a preferred embodiment the temperature is
selected from the rage of 0 to 140 °C. In a more preferred embodiment the temperature is
selected from the range of 60 to 130 °C. In the most preferred embodiment the temperature is
30 120 °C.
In an alternative embodiment the temperature during the sublimation process is varied during
the lyphilization process. In a preferred first embodiment the sublimation is carried out at two
temperatures. A schematic overview of preferred temperatures profile is shown in figure 3.
8
In one embodiment of the invention the sublimation process is carried out at two different
temperatures. Preferably the first temperature is selected from the range of -30 ° to + 40 °C and
the second temperature is selected from the range of 60 to 130 °C. In a preferred embodiment
the first temperature is selected from the range of -20 to 20 °C and the 5 second temperature is
selected from the range of 80 to 120 °C. In a most preferred embodiment the first temperature is
10 °C and the second temperature is 120 °C.
In an alternative embodiment the temperature profile comprises more than two different
10 temperatures. In an alternative embodiment the temperature profile comprises a continuous
temperature gradient.
In one embodiment of the invention the pressure during the sublimation step is between 50 μbar
and 800 μbar. In a preferred embodiment of the invention the pressure is between 75 μbar and
15 600 μbar, more preferably between 100 μbar and 400 μbar, even more preferably between 150
μbar and 300 μbar. In a most preferred embodiment the pressure during the sublimation step is
300 μbar.
The biopolymers produced with the process might be purified or isolated from the composition,
20 however it is preferred that no further purification or isolation step is performed. In the most
preferred embodiment the biopolymer is directly suitable for further processing and/or use.
The present invention does not only relate to a method for the production of biopolymers with
defined average molecular weight, but also to the use of said method for the production of
25 biopolymers with defined average molecular weight and to the biopolymers with defined average
molecular weight produced with said method.
In a preferred embodiment the method is used for the productions of biopolymers with defined
average molecular weight, which are selected from the group comprising hyaluronic acid,
30 collagen, glucosamino glycans, polysaccharides and fucoidanes. In a more preferred embodiment
the biopolymer is a glucosamino glycan. In a more preferred embodiment the method is used for
the productions of biopolymers with defined average molecular weight selected from alginates,
rhizobian gum, sodium carboxy methyl cellulose, pullulan, Biosaccharide Gum-1, glucomannane,
beta-glucane, pectine, tamarindus indica seed polysaccharide and hyaluronic acid. In an even
9
more preferred embodiment the the method is used for the productions of biopolymers with
defined average molecular weight selected from sodium alginate or hyaluronic acid. In the most
preferred embodiment the method is used for the production hyaluronic acid with a defined
average molecular weight.
5
In another aspect of the invention, the invention relates to a method for the production of
compositions, comprising a biopolymer, wherein the biopolymer has a defined average molecular
weight, the method comprising:
(i) providing a base composition comprising a biopolymer,
(ii) lyophilizing 10 said composition;
wherein the temperature maximum during the lyophilization process is selected to facilitate a
controlled and defined degradation of the biopolymer.
The inventors found that the present invention is suitable for the production of compositions,
15 comprising biopolymers. These compositions comprise a biopolymer with defined average
molecular weight and other optional components, such as dermatological, pharmaceutical or
cosmetic ingredients and just need to be emulsified or dissolved to be used.
In one embodiment of the invention the maximum temperature during the sublimation process is
20 selected from the range of – 40 °C to 150 °C. In a preferred embodiment the temperature is
selected from the rage of 0 to 140 °C. In a more preferred embodiment the temperature is
selected from the range of 60 to 130 °C. In the most preferred embodiment the temperature is
120 °C.
25 In an alternative embodiment the temperature during the sublimation process is varied during
the lyphilization process. In a preferred first embodiment the sublimation is carried out at two
temperatures. A preferred temperature profile is shown in figure 3.
In one embodiment of the invention the sublimation process is carried out at two different
30 temperatures. Preferably the first temperature is selected from the range of -40 ° to + 40 °C and
the second temperature is selected from the range of 60 to 130 °C. In a preferred embodiment
the first temperature is selected from the range of -20 to 20 °C and the second temperature is
selected from the range of 80 to 120 °C. In a most preferred embodiment the first temperature is
10 °C and the second temperature is 120 °C.
10
In one embodiment of the invention the pressure during the sublimation step is between 50 μbar
and 800 μbar. In a preferred embodiment of the invention the pressure is between 75 μbar and
600 μbar, more preferably between 100 μbar and 400 μbar, even more preferably between 150
μbar and 300 μbar. In a most preferred embodiment the pressure during 5 the sublimation step is
200 μbar.
In one embodiment of this aspect of the invention the biopolymer in the compositions is selected
from the group comprising hyaluronic acid, collagen, glucosamino glycans, polysaccharides and
10 fucoidanes. In a more preferred embodiment the biopolymer is selected from the group
consisting of alginates, rhizobian gum, sodium carboxy methyl cellulose, pullulan, Biosaccharide
Gum-1, glucomannane, beta-glucane, pectine, tamarindus indica seed polysaccharide and
hyaluronic acid. In an even more preferred embodiment the biopolymer is sodium alginate or
hyaluronic acid. In the most preferred embodiment the biopolymer is hyaluronic acid.
15
In a preferred embodiment the biopolymer is a biopolymer with high molecular weight. In a more
preferred embodiment the biopolymer is a biopolymer with native high molecular weight.
The composition comprising a biopolymer may comprise additional biopolymers or other
20 polymers. Any composition is suitable, as long as the composition comprises additionally water.
In a preferred embodiment the base composition is an aqueous solution or an emulsion
comprising a biopolymer.
25 In a more preferred embodiment the base composition comprises:
(i) the biopolymer,
(ii) water,
(iii) optionally one or more pharmaceutically, dermatologically and/or cosmetically acceptable
compounds and/or oils,
30 (iv) optionally an emulsifying agent
(v) optionally additional pharmaceutically, dermatologically and/or cosmetically active
components.
In alternative embodiments the base composition is a gel or a liquid with low to high viscosity.
11
The composition preferably contains further additional cosmetic, dermatological or
pharmaceutical ingredients or additions. Non-limiting examples for these ingredients are
emollients, cosmetically acceptable ingredients and dyes, perfumes or pharmaceutically active
5 substances like panthenol.
Non limiting examples for said ingredients or additions are: skin conditioning agents, skinsmoothing
agents, agents for skin hydration, e.g. panthenol or panthothenol, natural moisturising
factors, such as glycerine, lactid acid or urea. Alternatively a physical or chemical sunscreens,
10 keratolitics, such as α- or β-hydroxy acids, α- or β-ketoacids. Further possible ingredients include
radical catchers, anti-ageing agents, vitamins or derivatives thereof, e.g. vitamin C (ascorbic acid)
or esters or glycosides thereof, antioxidants, such as catechins or flavonoids.
Further potential ingredients comprise resveratrol, gluthation, ferulic acid, Q10, polyphenols,
15 ceramides, saturated and or unsaturated fatty acids and there glycerides. Furthermore esters,
such as wax esters, such as jojoba oil, triglycerides in general (neutral oil, argan oil, shea butter) or
unsaponifiable components from plant oils.
Further polysaccharides of vegetable, biotechnological or marine origin, as well as their
20 hydrolysates. Other ingredients might include enzymes, e.g. bromelain, coenzymes, enzyme
inhibitors, amino acids, natural and synthetic oligopeptides, peptides such as collagen and elastin,
as well as their hydrolysates, neuropeptides, growth factors, alcaloids. In some embodiments the
ingredients optionally include phytopharmaca such as aescin, ginsenosides, ruscogenine or aloin.
Further polymers are alginates, cellulose derivatives, starch, chitosan, chondroitin sulfate, further
25 synthetic biopolymers with biological function or compatibility
Non-limiting examples of cosmetic additions comprise skin lightening agents, inorganic or
synthetic fillers or decorative substances, such as coloring pigments or dyes or particles. Some
embodiments of the invention comprise substance for the cosmetic beautification of eyes, lips or
30 face.
In some embodiments the composition further comprises therapeutically active agents, such as
anti-acne or anti-rosacea agents, antimicrobial agents, such as silver and it’s derivatives, iodine or
PVP-iodine, antiperspirants, pain relieving substances such as lidocain or ibuprofen, adstringent
12
substances, deodorizing compounds, antiseborrhoeic substances or antiseptics. Furthermore cells
or cell components, such as autologous cells, allogenic cells, stem cells or platelet-rich plasma
(PRP).
The composition preferably contains other ingredients, e.g. stabilizers, 5 preserving agents, to
control the final parameters of the product, such a solubility or emulsifiability, mechanical
stability, product viscosity or haptics.
In a particular embodiment of the present invention the base composition is prepared and
10 provided in an appropriate container, which is suitable for the freezing and lyophilization process,
as well as optionally able to serve as packaging for the lyophilized composition comprising a
biopolymer with defined average molecular weight.
The invention further relates to the use of said method for the production of compositions
15 comprising a biopolymer with defined average molecular weight and to compositions comprising
biopolymers produced according to a method of the present invention.
In one embodiment of the present invention the final composition can serve as a basis for
aqueous liquids, emulsions with low viscosity, serum-like liquids, masks, creams, cream masks,
20 patches or segments for topical applications.
Figure Legends
Figure 1: Correlation of average molecular weight, skin penetration and biological activity of
25 hyaluronic acid.
Figure 2: Correlation of pH, temperature and obtained average molecular weight obtained, when
processing a composition comprising hyaluronic acid according to the present invention.
30 Figure 3: Overview of suggested temperature profiles over time during the lyophilization process.
Figure 4: Calibration curve to determine average molecular weight of hyaluronic acid, processed
according to the present invention.
13
Figure 5: Elugram and molecular mass profiles of neutral oil (5A) and Sepinov EMT-10 (5B)
processed according to the present invention.
Figure 6: comparison of the elution profiles (6A) and molecular mass profiles (6B) of native
hyaluronic acid and hyaluronic 5 acid, lyophilized 120 °C.
Figure 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.
10 Figure 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.
Figure 9: Molecular weight distributions of lyophilized samples of 1 wt-% high molecular weight
HA samples with pH adjusted to 6.21 and 2.9.
15
Figure 10: Molecular weight of hyaluronic acid containing emulsions lyophilized at different
process temperatures.
14
Examples
Example 1: Controlled degradation of hyaluronic acid
5
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 10 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.
15 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.
The following samples were analyzed:
20
1. Neutral oil, unprocessed
2. Sepinov EMT-10, unprocessed
3. Hyaluronic acid, unprocessed
4. Sepinov EMT-10, lyophilized at 120 °C
25 5. Hyaluronic acid, lyophilized at 120 °C
6. Mixture (hyaluronic acid, neutral oil and Sepinov EMT-10), lyophilized at 40 °C
7. Mixture, lyophilized at 60 °C
8. Mixture, lyophilized at 80 °C
9. Mixture, lyophilized at 100 °C
30 10. Mixture, lyophilized at 120 °C
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.
15
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 Figure 4).
Only pure hyaluronic acid samples were completely soluble. The soluble 5 components of the
Sepinov EMT-10 or neutral oil, do not produce any problematic signals during analysis (see
Figure 5 a and b).
The results clearly show that the composition and the lyophilization temperature affect the
10 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 Figures 6 a,b), the effect is stronger in the mixtures (see Figure 7 a,b).
Overall it is clearly visible that the choice of parameters during the lyophilization process is
15 suitable to control the average molecular weight of hyaluronic acid after the lyophilization.
Example 2: Influence of the pH-value on degradation
Hyaluronic acid with a molecular weight of 1.478 Mio Da (Contipro, Mw, according to gel
20 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.
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
25 according to the 10/120°C temperature profile shown in Figure 3.
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.
30
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 (Figure 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 Figure 8.
16
Example 3: Degradation of hyaluronic acids with different molecular weights
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-5 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.
7.5 ml HA solution was dispensed in 10 ml glass vials, samples were frozen at -20°C overnight and
10 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 Figure 3.
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
15 weight standards.
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.
20
Mw [kDa] nonprocessed
pH Mw [kDa]* processed at
10/120°C
Mw [kDa]* processed
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
17
Example 4: Emulsions containing hyaluronic acid
5g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in 465g
of distilled water, heated to 80°C and stirred by means of a Somakon MP-5 LB (1l) mixing device at
1400 rpm and ambient pressure for 15 minutes.
7.5g 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
10 μbar for further 15 minutes at 80°C.
25g of medium chain triglyciderides (MCTs) as model oil compound were added and homogenized
at 2100 rpm/200μbar for 5 minutes.
15 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.
Lyophilised samples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and
20 analyzed by means of GPC. Figure 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
25 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).
30 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 Figure 3.
18
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:
5
Mw [kDa]* nonprocessed
pH Mw [kDa] processed at
10/120°C
Mw [kDa] processed at
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:
Polymer Monomers pH Mw [kDa]*
nonprocessed
Mw
[kDa]**
processed
at
10/120°C
Mw [kDa]**
processed at
120°C
Rhizobian Gum tbd 5.59 706 533 592
3.50 706 354 nd
Sodium carboxy
methyl cellulose
Funktionalized
glucose
6.66 682 689 712
3.5 682 309 308
Pullulan Glucose
(Maltotriose)
5.46 314 239 278
3.50 314 61 nd
Biosaccharide
Gum-1
Fucose 7.35 2037**** 1735**** 1598****
3.50 2037**** 443 nd
19
Glucomannane Glucose, mannose 5.84 1304 1119 980
3.50 1304 247 nd
Beta-Glucan
(and) Pectin
Galacturonic acid,
rhamnose
4.02 778 389 358
Tamarindus
indica Seed
Polysaccharide
Glucose, xylose,
galoctoxylose
6.20 956 933 927
3.50 956 602 nd
Example 6: Lyophilisation of a hyaluronic acid emulsions containing different emulsifying polymer
components
3g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 5 1.5 MDa) was dissolved in
277.5g of distilled water heated to 80°C and stirred by means of a Somakon MP-LB (1l) mixing
device at 1400 rpm and ambient pressure for 15 minutes.
4.5g emulsifying polymer was added and the pH was adjusted to approximately 3 and mixture
10 was stirred at 1400 rpm/200 mbar for further 15 minutes at 80°C.
15g of medium chain triglyciderides (MCTs) were added and homogenized at 2100 rpm/200mbar
for 5 minutes.
15 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.
The following table shows the emulsifying polymer used, the pH values as well as the viscosity of
20 the emulsion (measured with a hand-held HAAKE Viscotester 2 plus) prior to lyophilisation. All
freeze-dried samples provided fast rehydration to opaque emulsions.
20
Example 7: Lyophilisation of a hyaluronic acid emulsions containing different oil components
5
3g of high molecular weight HA (GfN/Contipro 3010, 1.5 MDa), 277.5g of distilled water, 4.5g
EMT-10 as well as 15g of oil component (MCT (Cosnaderm), Marula Oil (Seatons), Jojobaoil (J.H.
Müller GmbH) or Argan oil (Seatons)) were processed as described in example 6.
7.5 ml of the resulting emulsions was dispensed in 10 ml glass vials, samples 10 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.
15 Example 8: Lyophilisation of a hyaluronic acid emulsions containing a UV filter blend
3g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in
277.5g of distilled water heated to 80°C and stirred by means of a Somakon MP-LB (1l) mixing
device at 1400 rpm and ambient pressure for 15 minutes. 4.5g Sepinov EMT-10 was added and
20 mixture was stirred at 1400 rpm/200 mbar for further 15 minutes at 80°C.
6g Eusolex 9010 (Avobenzone), 15g Eusolex OCR (Octocrylene) and 7.5g Eusolex OR (Ethylhexyl
Salicylate) were dissolved in 15g of medium chain triglyciderides (MCTs) and the UV filter mixture
was added to the polymer solution and homogenized at 2100 rpm/200mbar for 5 minutes.
25
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.
21
Example 9: Lyophilisation of a hyaluronic acid emulsions containing microcrystalline silver
3g of high molecular weight hyaluronic acid (GfN/Contipro 3010, 1.5 MDa) was dissolved in
277.5g of distilled water heated to 80°C and stirred by means of a Somakon 5 MP-LB (1l) mixing
device at 1400 rpm and ambient pressure for 15 minutes.
4.5g 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 15g of medium chain
10 triglyciderides (MCTs) and the mixture was added to the polymer solution and homogenized at
2100 rpm/200mbar for 5 minutes.
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
15 shown in Fig. 3. All freeze-dried samples provided fast rehydration resulting in a colorless gel.
22
We claim:
1. Method for the production of a biopolymer, wherein the biopolymer has a defined
average molecular weight, the method comprising
lyophylizing a composition comprising 5 the biopolymer,
optionally purifying and/or isolating the biopolymer;
wherein the maximum temperature during the lyophilization process is selected to
facilitate a controlled and defined degradation of said biopolymer.
10 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 any of the claims 1 or 2, wherein the composition comprising
the biopolymer has a defined pH value.
15
4. The method according to claim 3, wherein the pH value is selected from a range between
1.5 and 8.5, preferably between 2.5 and 6.
5. The method according to any of the preceding claims, wherein the temperature during
20 the sublimation process is selected from a range between -40 °C and 150 °C.
6. The method according to any of the preceding claims, wherein the pressure during the
sublimation process is selected from a range between 50 μbar and 800 μbar.
25 7. The method according to any of the preceding claims, wherein the biopolymer is selected
from the group comprising hyaluronic acid, collagen, glucosamino glycans,
polysaccharides and fucoidanes, preferably the biopolymer is hyaluronic acid.
8. A method for the production of compositions, comprising a biopolymer, wherein the
30 biopolymer has a defined average molecular weight, the method comprising:
(i) providing a base composition comprising a biopolymer with native high molecular
weight,
(ii) lyophilizing said base composition;
23
wherein the maximum temperature during the lyophilization process is selected to
facilitate a controlled and defined degradation of the biopolymer.
9. The method according to claim 8, wherein the base composition is an emulsion or an
5 aqueous solution.
10. The method according to claim 9, wherein the base composition is an emulsion
comprising:
(i) the biopolymer,
10 (ii) water,
(iii) optionally a pharmaceutically, dermatologically or cosmetically acceptable oil,
(iv) optionally an emulsifying agent and
(v) optionally emollients.
15 11. The method according to claim 10, wherein the base composition additionally comprises
further dermatological, pharmaceutical or cosmetic additions.
12. The method according to any of the claims 8 to 11, wherein the base composition
comprises additional components, so that the resulting lyophilized product is easily
20 dissolvable or easily forming an emulsion.
13. The method according to any of the claims 8 to 12, wherein the base composition is
prepared in an appropriate container, which is suitable for the lyophilization process and
which can serve as primary packaging.
25
14. The method according to any of claims 8 to 13, wherein the biopolymer is selected from
the group comprising hyaluronic acid, collagen, glucosamino glycans, polysaccharides and
fucoidanes, preferably the biopolymer is hyaluronic acid.
30 15. Use of a method according to any of the claims 1 to 7 for the production of a
pharmaceutically, dermatologically or cosmetically acceptable biopolymer.
16. Use of a method according to claims 8 to 14 for the production of pharmaceutically,
dermatologically or cosmetically acceptable compositions comprising a biopolymer.
24
17. A composition produced according to any of the claims 8 to 14.
18. Use of a composition according to claim 17 as a pharmaceutical, dermatological or
5 cosmetic product.
19. Use of a biopolymer produced by a method according to claims 1 to 7 or a composition
according to claim 17 for the production of a pharmaceutical, dermatological or cosmetic
product.