METHODS FOR THE PRODUCTION OF BIOPOLYMERS WITH SPECIFIC MOLECULAR WEIGHT DISTRIBUTION
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
applica ble su bstances and in the use and application thereof.
BACKGROUND
Biopolymers, such as collagen, polysaccharides or hyaluronic acid are commonly used in cosmetic
or dermatological compositions. In many cases these biopolymers are used as moisturizers or ant i
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
human tissue. It is an anionic, non-sulfated glycosaminoglycan comprising the following structure:
Hyaluronic acid has several medical uses, in particu lar in dermatology, and is commonly used in
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
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.
EP 2 479 194 A2 describes the hydrolysis of hyaluronic acid on activated charcoal. EP 2 463 309 Bl
and EP 1 992 645 Al describe several methods for the acidic hydrolysation of hyaluronic acid. Other
methods involve the use of enzymatic hydrolysis and filtration (EP 0 138 572 Bl) or the use of high
temperatures and strong shearing forces (EP 1 987 153 Bl).
The problem with all these methods is, that in particular hyaluronic acid needs extensive
purification steps t o remove the low molecular weight hyaluronic acids, which can be pro
inflammatory.
It is therefore necessary to provide a method for the efficient prod uction 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.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to 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 and a defined molecular weight distribution, the method comprising
(i) 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 compositions in a reaction vessel without su bstantially mixing each
other;
(iv) optionally freezing the compositions;
(v) lyophylizing the compositions comprising the biopolymers,
(v) optionally purifying and/or isolating the biopolymers;
wherein the maximu m temperature during the lyophilization process is selected t o facilitate a
controlled and defined degradation of said biopolymer and wherein the pH-values of the first and
second composition differ by at least 0.1 or wherein the first and second composition comprise
different biopolymers.
In one embodiment of the invention the biopolymers are biopolymers with high molecular weight.
In a preferred embodiment the biopolymers are biopolymers with native high molecular weight.
In a preferred embodiment the invention relates to 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 and a defined weight distribution, the method comprising
(i) providing a first composition comprising a biopolymer with high molecular weight
with a defined pH-value between 1.5 and 8.5;
(ii) providing a second composition comprising a biopolymer with high molecular
weight with a defined pH-value between 1.5 and 8.5
(iii) combining the compositions in a reaction vessel without su bstantially mixing these
with each other;
(iv) optionally freezing the compositions;
(v) lyophylizing the compositions comprising the biopolymers,
(vi) optionally purifying and/or isolating the biopolymers;
wherein the temperature during the lyophilization process is selected to facilitate a controlled and
defined degradation of said biopolymer and wherein the pH-values of the first and second
composition differ by at least 0.1 or wherein the first and second composition comprise different
biopolymers.
In one preferred embodiment of the invention the biopolymers are biopolymers with high
molecular weight. In a more preferred embodiment the biopolymers are biopolymers with native
high molecular weight.
The invention further relates to the use of said method for the production of biopolymer
compositions and to biopolymer compositions, which are produced by said method.
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
prefera bly not technically or chemically modified, besides the common and native modifications,
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
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 t o polypeptides.
Polymers consisting of biological units, but not produced in a living organism, such as polylactic
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.
Non-limiting examples for biopolymers accord ing to the present invention comprise: collagens,
starch, cellulose derivatives, glucosamino glycans, polysaccarides or fucoidanes.
In the context of the present invention a frozen composition refers to a composition, which is in a
solid state of matter, rega rdless of its state of matter at 25 °C. In most embodiments a frozen
composition is a composition in a liquid state of matter at 25 °C, which has been cooled down to a
solid state of matter. In one embodiment cooling is done by shock-freezing in liquid nitrogen.
Alternative ways of cooling are cooling and freezing in a freezer. In a preferred emboidment
freezing is done at a temperature between -50 °C and -4 °C.
In the context of the present invention lyophilization or lyophilizing refers t o a dehydration process,
wherein water is removed by su blimation. Lyophilization is commonly referred to as freeze drying.
In general lyophilization comprises three stages:
(i) freezing the composition t o be dehydrated, wherein it is important that the composition is
cooled below its triple point. The suita ble freezing method is dependent on the 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 m bar or even lower. In this stage the temperature is usually adjusted to
get the water su blimated. Prefera bly, but not necessarily, at this stage the temperature
remains under 0°C.
(iii) a secondary drying phase, wherein the pressure is optionally reduced, down t o the range
of mbars and the temperature prefera bly raised above 0 °C to remove more strongly bound
water.
In one embodiment of the invention, the temperature is controlled during the second drying 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.
Within the meaning of the present invention the "temperature during the su blimation process"
refers t o 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 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-solu ble biopolymers or non-solu ble
cosmetic additions in water.
In the context of the present invention the term emulsion refers to mixtures of normally immiscible
liqu ids. In the context of the present invention the term emulsion in particular refers t o water-inoil
or oil-in-water emulsion. Prefera bly in the context of the present invention the emulsion is
sta bilized by the use of an emulsifying agent or emulsifier. Non-limited examples for emulsifying
agents are lecithin, sodium stearoyl lactylate, polymers with emulsifying functionalities or
detergents.
In the context of the present invention a reaction vessel is any suita ble vessel for containing and
processing the compositions. Prefera bly said vessel is suita ble for freezing and lyophilization
processes.
DETAILED DESCRIPTION OF THE INVENTION
The inventors found surprisingly, that biopolymers can be su bjected to controlled degradation
during lyophilization processes, resulting in biopolymers with defined average molecular weight. In
add ition the inventors found, that the molecular weight distribution of the degraded biopolymer
can be influenced by combining frozen compositions during the lyophilization process.
A first aspect of the present invention relates to 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 and a defined weight distribution, the method comprising
(i) 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 compositions in a reaction vessel without su bstantially mixing these
with each the compositions;
(iv) optionally freezing the compositions;
(v) lyophylizing the compositions comprising the biopolymers,
(vi) optionally purifying and/or isolating the biopolymers;
wherein the maximum temperature during the lyophilization process is selected t o facilitate a
controlled and defined degradation of said biopolymer and wherein the pH-values of the first and
second composition differ by at least 0.1 and/or wherein the first and second composition comprise
different biopolymers.
Combining the compositions without su bstantially mixing the compostions refers t o a process,
wherein the compositions are com bined in one reaction vessel but retain individual concentrations
of compounds and other properties, such as pH value. This could be done by providing and
combining two frozen compositions or by combining two compositions with high viscosity by
overlaying the compositions and freezing them prior t o lyophilization.
In a preferred embodiment the compositions are frozen and then com bined in one reaction vessel.
In a preferred embodiment the biopolymers are biopolymers with high molecular weight. In a more
preferred embodiment the biopolymers are biopolymers with native high molecular weight.
The compositions comprising a biopolymer can be any kind of composition, provided said
compositions comprise at least small amounts of water in addition to said biopolymer. Said
composition may comprise additional biopolymers, i.e. mixtures.
The first and second compositions might comprise different biopolymers. In a preferred
embodiment both compositions comprise the same biopolymer. In another preferred embodiment
the first and second composition comprise only one biopolymer each.
In a preferred embodiment the biopolymers are biopolymers with high molecular weight. In a more
preferred embodiment the biopolymers are biopolymers with native high molecular weight.
The method is in particular suita ble for biopolymers selected from the group comprising hyaluronic
acid, collagen, glucosamino glycans, polysaccharides and fucoida nes. In a preferred embodiment
the biopolymer is a glucosamino glycan or polysaccarid. In a more preferred embodiment the
biopolymer is selected from the grou p 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.
In a preferred embodiment the first and/or second composition comprising the at least one
biopolymer is an aqueous solution or an emulsion. In a more preferred embodiment both
compositions comprising a biopolymer are aqueous solutions or emulsions.
In another embodiment of the present invention the first and/or second composition comprising a
biopolymer is a gel or a liquid with low t o high viscosity.
The inventors had fou nd in particular, that controlled conditions during the su blimation process
and a control of the parameters of the compositions, e.g. salt contents, pH-value, vacuum, used
emulsifying agents, allow the control of the average molecular weight of the degraded biopolymer.
In particular the inventors found that com bining compositions comprising the same biopolymer
with different pH-values leads t o a different distribution of the average molecular weight of the
biopolymer, which allows differently defined weight distributions depending on the pH-values
and/or volume of the compositions.
In one embodiment of the invention the first and/or second frozen composition comprising a
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.
If the first and second composition comprise the same biopolymer it is preferred that the pH value
of the composition differs by at least 0.1. In a preferred embodiment the pH-value of first and
second composition differs by at least 0.5. In a more preferred embodiment the pH-value of first
and second composition differs by at least 1. In the most preferred embodiment the pH value of
the first and second frozen composition differs by at least 2.
The inventors had found a direct correlation between the pH-value, the temperature during the
su blimation 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 com bination of temperature and pH value selected. The inventors
further fou nd that it is possible to stack or mix multiple frozen compositions to vary the molecular
weight distribution of the final product.
In one embodiment of the invention the maximum temperature during the su blimation 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 120 °C.
In an alternative embodiment the temperature during the su blimation process is varied during the
lyphilization process. In a preferred first embodiment the su blimation is carried out at two
temperatures. A schematic overview of preferred temperatures profile is shown in figure 3.
In one embodiment of the invention the su blimation process is carried out at two different
temperatures. Prefera bly 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 t o 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.
In an alternative embodiment the temperature profile comprises more than two different
temperatures. In an alternative embodiment the temperature profile comprises a continuous
temperature gradient.
In one embodiment of the invention the pressu re during the su blimation step is between 50 mbar
and 800 m bar. In a preferred embodiment of the invention the pressure is between 75 mbar and
600 mbar, more prefera bly between 100 m bar and 400 mbar, even more prefera bly between 150
m bar and 300 mbar. In a most preferred embodiment the pressure during the su blimation step is
300 m bar.
The biopolymer compositions prod uced with the process might be purified or isolated from the
composition, however it is preferred that no further purification or isolation step is performed. In
the most preferred embodiment the biopolymer is directly suita ble for further processing and/or
use.
The present invention does not only relate t o a method for the production of biopolymer
compositions with defined average molecular weight, but also t o the use of said method for the
production of biopolymer compositions with defined average molecular weight and to the
biopolymer compositions 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 grou p comprising hyaluronic acid, collagen,
glucosamino glyca ns, 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 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.
The inventors found that the present invention is suita ble for the production of complex
compositions, comprising biopolymers. These compositions comprise at least one 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.
The complex compositions may comprise additional biopolymers or other polymers. Any
composition is suita ble, as long as the composition comprises additionally water.
In a preferred embodiment the first and/or second composition comprises:
(i) a biopolymer,
(ii) water,
(iii) optionally a pharmaceutically, dermatologically and/or cosmetically accepta ble compounds
and/or oils;
(iv) optionally an emulsifying agent
(v) optionally additional pharmaceutically, dermatologically or cosmetically active
components.
In alternative embodiments the first and/or second frozen composition is a gel or a liquid with low
t o high viscosity.
The first and/ or second composition prefera bly contains further additional cosmetic,
dermatological or pharmaceutical ingredients or additions. Non-limiting examples for these
ingredients are emollients, cosmetically accepta ble ingredients and dyes, perfumes or
pharmaceutically active su bstances like panthenol.
In a preferred embodiment the first and second frozen composition are comprise the same
compounds with the exception of the biopolymer or the pH-value. In the most preferred
embodiment the first and second frozen composition comprise the same compounds and the same
biopolymer and differ only in pH-value.
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,
keratolitics, such as a- or b-hydroxy acids, a- or b-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 resveratol, gluthation, ferulic acid, Q10, polyphenols,
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 ingredients comprise polysaccharides of vegeta ble, biotechnological or marine origin, as
well as their hydrolysates. Other ingred ients 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 synthetic biopolymers with biological function or compatibility
Non-limiting examples of cosmetic additions comprise skin lightening agents, inorganic or synthetic
fillers or decorative su bsta nces, such as coloring pigments or dyes or particles. Some embodiments
of the invention comprise su bstance for the cosmetic beautification of eyes, lips or face.
In some embodiments the first and/or second frozen 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 su bstances such as lidocain or
ibuprofen, adstringent su bstances, deodorizing compounds, antiseborrhoeic su bstances or
antiseptics. Furthermore cells or cell components, such as autologous cells, allogenic cells, stem
cells or platelet-rich plasma (P P).
The first and/or second composition prefera bly contains other ingredients, e.g. sta bilizers,
preserving agents, to control the final parameters of the product, such a solu bility or emulsifia bility,
mechanical sta bility, product viscosity or haptics.
In a particular embodiment of the present invention the first and second composition are combined
in an appropriate container, which is suita ble for the freezing and lyophilization process, as well as
optionally able t o serve as packaging for the lyophilized composition comprising at least one
biopolymer with defined average molecular weight and defined weight distribution.
The invention further relates t o the use of said method for the production of compositions
comprising a biopolymer with defined average molecular weight and t o 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
liqu ids, emulsions with low viscosity, serum-like liquids, masks, creams, cream masks, patches or
segments for topical applications.
FIGURE LEGENDS
Figure 1 : Correlation of average molecular weight, skin penetration and biological activity of
hyaluronic acid.
Figure 2 : Correlation of pH, temperature and obtained average molecular weight obtained, when
processing a composition comprising hyaluronic acid according t o the present invention.
Figure 3 : Overview of suggested temperature profiles over time during the lyophilization process.
Figure 4 : Calibration curve t o determine average molecular weight of hyaluronic acid, processed
according to the present invention.
Figure 5 : Elution and molecular mass profiles of neutral oil (a) and Sepinov EMT-10 (b) processed
according to the present invention.
Figure 6 : comparison of the elution profiles (A) and molecular mass profiles (B) of native hyalu ronic
acid and hyalu ronic acid, lyophilized 120 °C.
Figure 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.
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.
Figure 10: Molecular weight of hyaluronic acid containing emulsions lyophilized at different process
temperatures.
Figure 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
Figure 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
Figure 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
Deionized water is transferred to 1 I 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 mbar).
The oil component is added and stirred at 1400 rpm/45 °C/200 mbar for 10 min and su bsequently
for 5 min at 2100 rpm/45 °C/200 mbar. 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 su bsequently lyophilized up t o maximum target temperature.
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 :
1. Neutral oil, unprocessed
2. Sepinov EMT-10, unprocessed
3. Hyaluronic acid, unprocessed
4. Sepinov EMT-10, lyophilized at 120 °C
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
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-solu ble parts were
removed by filtration.
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 solu ble. The solu ble 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 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 su ita ble
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
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
according to the 10/120°C temperature profile shown in Figure 3.
Lyophilised sa mples 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.
Independent on adjusted pH, all samples were cleaved showing a maximu m 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.
Example 3: Degradation of hyaluronic acids with different molecular weights
Four differents types of hyaluronic acid (Contipro/GfN 3010 (MW: 1478 kDa), Principium Cu be3
(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.
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 Figure 3.
Lyophilised sa mples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed
by means of gel permeation chromatography against Pu llulan and Dextran molecular weight
standards.
High and medium molecular weight hyalu ronic acid showed a moderate decay of molecular weight
at original pH dissolved in distilled water, whereas molecular weight of su bstances decayed
drastically at low pH, as shown in the following t able.
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-LB (II) mixing device at 1400
rpm and ambient pressu re 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 mbar
for further 15 minutes at 80°C.
25g of mediu m chain triglyciderides (MCTs) as model oil compound were added and homogenized
at 2100 rpm/200 m bar for 5 minutes.
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 analyzed
by means of GPC. Figure 9 shows the temperatu re dependence of the molecular weight (Mw) of
the hyaluronic acid decreasing with increasing maximum process temperature.
Example 5: Lyophilization of different biopolymers
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 t o 0.3 wt-% in PBS buffer (pH 7.4).
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.
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:
Polysaccha
Polymer Monomers pH Mw [kDa]* Mw Mw [kDa]**
non- [kDa]** processed at
processed processed 120°C
at
10/120°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
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 t o pH 2.9.
pH 6.21 HA solution was dispensed in differed volu mes 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 t o 0.75 ml and frozen
again and stored at -20°C overnight. The corresponding volume ratios are shown in the following
t able:
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.
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.
Dependant on volume ratio differently shaped molecular weight distru butions 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 achived. Dependant on targeted
biological function an optimum molecular weight distribution can be designed.
Example 7: pH and volume variation with stacked pullulan solutions
Pullulan with a molecular weight of 371 kDa (Hayashibara, Mw, according to gel permeation
chromatogra phy) 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 t o pH 3.5.
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 t o 0.75 ml and frozen
again and stored at -20°C overnight. The corresponding volume ratios are shown in the following
table.
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.
Lyophilised sa mples were diluted in GPC buffer (pH 7.4) at a concentration of 0.3 wt-% and analysed
by means of gel permeation chromatography against Pu llulan and Dextran molecular weight
standards.
Dependant on volume ratio differently shaped molecular weight distru butions 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
Sodium alginate with a molecular weight of 881 kDa (Cargill, Mw, according t o gel permeation
chromatogra phy) 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 t o pH 3.5.
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.
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.
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 molelecur weight is mainly affected by the lyophilisation conditions
and less by the amount of the low pH solution.

CLAIMS
1. 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
(i) providing a first frozen composition comprising a biopolymer with a defined pHvalue;
(ii) providing a second frozen composition comprising a biopolymer with a defined pHvalue
(iii) combining the compositions in a reaction vessel without su bstantially mixing these
with each other;
(iv) optionally freezing the compositions;
(v) lyophylizing the compositions comprising the biopolymers,
(v) optionally purifying and/or isolating the biopolymers;
wherein the maximum temperature during the lyophilization process is selected to
facilitate a controlled and defined degradation of said biopolymer and wherein the pHvalues
of the first and second composition differ by at least 0.1 and/or wherein the first and
second composition comprise different biopolymers.
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 any of the claims 1 to 3, wherein the first and/or second
composition comprising a biopolymer is a frozen aqueous solution or an emulsion.
5. The method according to any of the claims 1 to 4, wherein the pH value of the first and/or
second composition is selected from a range between 1.5 and 8.5, prefera bly between 2.5
and 6.
6. The method according to any of the preceding claims, wherein the temperature during the
su blimation process is selected from a range between -40 °C and 150 °C.
7. The method according to any of the preceding claims, wherein the pressure during the
su blimation process is selected from a range between 50 mbar and 800 mbar.
8. The method according to any of the preceding claims, wherein the biopolymer of the first
and/or second composition is selected from the group comprising hyaluronic acid, collagen,
glucosamino glycans, polysaccharides and fucoidanes, preferably the biopolymer is
hyaluronic acid.
9. The method according to claims 1 to 8, wherein the first and/or second composition is an
emulsion comprising:
(i) a biopolymer,
(ii) water,
(iii) optionally a pharmaceutically, dermatologically and/or cosmetically accepta ble
compound or oil,
(iv) optionally an emulsifying agent and
(v) optionally emollients.
10. The method according to any of claims 1 to 9, wherein the first and/or second composition
additionally comprises further dermatological, pharmaceutical or cosmetic additions.
11. The method according to any of the claims 1 to 10, wherein the first and/or second
composition comprises additional components, so that the resulting lyophilized product is
easily dissolva ble or easily forms an emulsion.
12. The method according to any of the claims 1 to 11, wherein the first and second
composition comprise the same contents and differ only in pH-value and/or the
biopolymer.
13. The method according to any of the claims 1 to 12, wherein the first and second
composition are combined in an appropriate container, which is suita ble for the
lyophilization process and which can serve as primary packaging.
14. The method according to any of claims 1 to 13, wherein the biopolymer of the first and/or
second composition is selected from the group comprising hyaluronic acid, collagen,
glucosamino glycans, polysaccharides and fucoidanes, preferably the biopolymer is
hyaluronic acid.
15. Use of a method according to any of the claims 1 to 14 for the production of a
pharmaceutically, dermatologically or cosmetically accepta ble biopolymer composition.
16. A composition produced according to any of the claims 1 to 14.
17. Use of a composition according to claim 16 as a pharmaceutical, dermatological or cosmetic
product.
18. Use of a biopolymer composition according to claim 16 for the production of a
pharmaceutical, dermatological or cosmetic product.