|Title of Invention||
PROCESS FOR PREPARING A STERILE HIGH MOLECULAR WEIGHT HYALURONIC ACID FORMULATION
|Abstract||A process for preparing a sterile ready-to-use aqueous pharmaceutical formulation comprises a high molecular weight hyaluronic acid salt (HA) at a specified concentration, comprising the steps of: providing an aqueous formulation comprising high molecular weight HAat a concentration of less than the specified final concentration; passing said aqueous formulation through a filter having a pore sizeless than 0.45 pm; concentrating said aqueous formulation by applying a vacuum and boiling off water until said specified concentration is reached.|
|Full Text||PROCESS FOR PREPARING A STERILE HIGH MOLECULAR WEIGHT HYALURONIC ACID FORMULATION
The present invention relates to a process for preparing a sterile high molecular
weight hyaluronic acid salt as a final formulation for pharmaceutical use.
Hyaluronic acid in its salt form may for instance include sodium hyaluronate,
potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or
Hyaluronic acid is a mucoid polysaccharide of biological origin, which is widely
distributed in nature. For example, it is known that hyaluronic acid is present in
various animal tissues such as umbilical cord, synovial fluid, vitreous humor,
rooster comb and various connective tissues such as skin and cartilage.
Chemically, hyaluronic acid is a member of glycosaminoglycans and it is
constituted by alternating and repeating units of D-glucuronic acid and N-acetyl-
D-glucosamine, to form a linear chain having a molecular weight up to 13 x 106
In the meaning of the present invention, high molecular weight hyaluronic acid
is hyaluronic acid having an average molecular weight of not less than 0.5 x 106
Pharmaceutical use of hyaluronic acid or of a salt thereof is widely described in
Since hyaluronic acid is a non-immunogenic substance and has viscoelastic
and hydrophilic properties, it is used, since several years, as an eye vitreous or
joint fluid replacement or as a supportive medium in ophthalmic surgery, as
disclosed for example in US-A-4,141,973 of Balazs.
In joint fluids, the viscous hyaluronic acid solution serves as a lubricant to
provide a protective environment to the cells, and for this reason, it is used in
the treatment of inflamed knee joints.
EP-A-0 781 547 discloses a sodium hyaluronate based ophthalmic formulation
for use in eye surgery.
EP-A-0 719 559 discloses sodium hyaluronate viscous solutions for use as
masking fluid in therapeutic photokeratectomy by means of excimer laser.
EP-A-0 875 248 discloses the use of hyaluronic acid or of one of its
pharmaceutically acceptable salts for the preparation of an aqueous solution
useful as intra-articular lavage liquid.
EP-A-0 698 388 of Chemedica S.A. discloses an ophthalmic preparation for
use as artificial tears containing hyaluronate as a viscosity thickener.
The pharmaceutical use of hyaluronic acid or of a salt thereof requires a highly
pure and sterile product.
Hyaluronic acid can be extracted and purified from animal or microbial sources
such as umbilical cords, rooster combs or from group A and C Streptococci as
disclosed for example in US-A-4,141,973 of Balazs, US-A-5,559,104 of Romeo
et al. and WO 00/4925.
Industrial extraction and purification processes of hyaluronic acid typically
produce hyaluronic acid salts, such as sodium hyaluronate, in the form of a
dried powder. The purified pharmaceutical grade dried povfer may be used for
preparing, for example, aqueous pharmaceutical formulations for the various
pharmaceutical uses such as interarticular injection, eye drops or vitreous
A common industrial process for preparing ready-to-use pharmaceutical
formulations comprises the mixing of a defined quantity in weight of sodium
hyaluronate with a precise volume of water and, as the case may be, salt such
as sodium chloride and buffers such as phosphates and other excipients. As
the concentration and composition of the formulation for pharmaceutical use
should remain within a narrowly defined range, the various components of the
formulation are carefully measured. The formulation is then filled into recipients
such as syringes and vials of defined dosages ready for use. Subsequent to
filling of the recipients, the formulation is sterilized by autoclave typically at
around 121° C for fifteen minutes or more.
One of the problems with the use of heat to sterilize hyaluronic acid is the
known effect on breaking the molecular chains forming HA, thus reducing the
average molecular weight of HA.
The high molecular weight of hyaluronic acid is an important pharmacological
In many pharmaceutical applications it is undesirable to have low molecular
weight hyaluronic acid in the formulation, for example in view of the
inflammatory effects of low molecular weight HA as reported in US 4,141,973
and the loss of beneficial rheological properties of high molecular weight HA. In
order to compensate for degradation of the HA in a formulation of given
concentration in the aforementioned sterilization methods, the hyaluronic acid
or salts thereof initially used in preparing the formulation have an. average
molecular weight that is higher than that of the desired minimum average
molecular weight of the final formulation. This is however uneconomical since
the yield of hyaluronic acid from starting material decreases as the average
molecular weight required increases..
Another known method of sterilizing hyaluronic acid is by filtration. This
technique is used in conventional industrial processus for preparing purified
hyaluronic acid salts in a concentrated form, usually in the form of dried
powder, whereby a low concentration aqueous solution is passed through the
filter and subsequently dried.
Such sterilization steps are for example described in European patent
application EP 867453 and in PCT application WO 00/44925. In these
applications, a filter with a pore size as small as 0.22 µm is also disclosed for
sterilization: Filters having a pore size of 0.22 µm have a bacterial challenge of
1 over 107 bacteria, based on the smallest known bacterium Pseudomonas
diminuta, while filters having a pore size of 0.45 µm have a bacterial challenge
of 1 over 104 bacteria (always based on Pseudomonas diminuta). For this
reason, filters having a pore size less than 0.45 µm are considered to be
In conventional industrial processes, the method of sterilization by filtration is
not known to be used for preparing high viscosity pharmaceutical formulations
ready for use, since at the required concentration of HA in high viscosity
pharmaceutical aqueous formulations, typically in the range of 1 .to 2% wt/v, not
all of the hyaluronic acid passes through the filter at 0.22 µm. Since this results
in a change in the concentration and/or a loss of hyaluronic acid, sterilization by
filtration for preparing ready-to-use high viscosity pharmaceutical formulations
In US 5,093,487, the preparation of an aqueous pharmaceutical formulation
comprising high molecular weight sodium hyaluronate ready for use and
sterilized through a filter of 0.22 µm is described. The sterilizing method
described in this patent however relies on a number of passes of hyaluronic
acid aqueous formulation through the 0.22 pm filter, so as to irreversibly reduce
the viscosity of the hyaluronic acid. The sterilization of an aqueous
pharmaceutical formulation comprising HA at a concentration of 1% or more is.
according to this publication, possible in view of the reduction of viscosity of the
hyaluronic acid resulting from the multiple passes through the filter. It is further
argued in this application that the viscosity is reduced without reducing the
molecular weight of HA. Without wishing to take position on the validity of the
findings reported in the aforementioned publication, for many pharmaceutical
applications such as intra-articular applications, the lowering of the viscosity of
HA is undesirable.
An object of the present invention is to obtain a sterile ready-to-use
pharmaceutical aqueous formulation comprising a hyaluronic acid salt, that is
sterile and economical to produce, particularly in industrial conditions. It is
advantageous to provide such formulation with a narrow tolerance in the
concentration of the ingredients of the formulation.
Objects of this invention have been achieved by a process for preparing a
sterile high molecular weight hyaluronic acid formulation for pharmaceutical use
according to claim 1.
Disclosed herein is a process for preparing a sterile ready-to-use aqueous
pharmaceutical formulation comprising a high molecular weight hyaluronic acid
salt (HA) at a specified final concentration, comprising the steps of:
- providing an aqueous formulation comprising high molecular weight HA at a
concentration less than the specified final concentration;
- passing said aqueous formulation through a filter having a pore size
not exceeding 0.45 urn;
- concentrating said aqueous formulation by applying a vacuum and boiling off
water until said specified final concentration is reached.
Advantageously, the reduced concentration of the aqueous formulation prior to
filtering, as a function of the molecular weight, reduces the viscosity and
enables the entire HA to pass through the filter and be sterilized, the
subsequent boiling with a vacuum ensuring that essentially no heat dependent
degradation of HA occurs. The process is well adapted to an industrial
environment and is particularly economical, allowing ready-to-use dosages of
pharmaceutical formulation comprising HA to be filled in sterile recipients
without further sterilization.
A further advantage is that the formulation is microbiologically stable and may
be kept for weeks before being used as pharmaceutical preparation.
Viscosity of HA in aqueous solution is a property that depends on several
parameters such as molecular weight, concentration, temperature,
concentration and quality of salts, pH and shear rate applied to the solution.
Higher molecular weight and concentration increase the viscosity, while higher
shear rate and salts decrease the viscosity. Regarding the temperature, HA has
a hysteretic behavior as reported by M. Cardones et al. "Hysteresis Behavior of
Sodium Hyaluronate Solutions during Heating and Cooling", Clear Solutions
Biotech, Inc., Technical Rep 01. Viscosity increases and decreases in an
irregular way by increasing or decreasing the temperature. Between 60° C and
70° C it is possible to get the minimum viscosity of the solution. Therefore, this
hysteretic property can be used for decreasing or increasing the viscosity of the
solution, but it has to be considered that the HA chains degradation is
proportional to the temperature and the duration at which this temperature is
During the concentrating step after filtration, the concentration of HA may be
monitored in real time in order to stop the vacuum boiling when the specified
concentration for the ready-to-use pharmaceutical formulation is reached. The
monitoring or measuring process may advantageously be carried out with a
spectrophotometer with the sensing beam placed in the formulation, the
absorption of radiation in the ultraviolet range (UV) being proportional to the HA
concentration. A particularly advantageous feature of this invention is that it
obviates the need to mix exact quantities of water and hyaluronic acid to obtain
the specified concentration and ensure that such concentration is maintained
through the process. Instead, the initial HA and water mix has an approximate
concentration lower than the final formulation, thus simplifying the process.
The vacuum applied during the concentrating process is preferably j not exceeding
200 millibars absolute pressure, in particular in the range of 30 to 60 millibars,
for example 40 millibars, whereby the boiling temperature is around 26 to 28°
C. Industrial equipment is economical to operate reliably at such pressures, and
the low temperature avoids any significant or measurable reduction of the
hyaluronic acid molecular weight.
The filter pore size is advantageously not exceeding 0.22 urn, thus ensuring the
preparation of a highly sterile formulation. Filter sterilized ready-to-use high
viscosity pharmaceutical formulation with HA concentration in the range of 1 to
3% can thus advantageously be prepared in a sterile and economical manner,
according to this invention.
Further advantageous aspects of this invention will be apparent from the claims
and the following detailed description of an example of a process and annexed
drawing in which:
Fig. 1 represents a flow chart illustrating steps in an embodiment of the process
according to the invention;
Figures 2 and 3 are graphs represent the percentage of HA of molecular weight
1.1 million and 2.2 million Daltons, respectively, passing through a filter of 0.22
urn as a function of the % wt/v concentration in an aqueous solution, at 20° C
and neutral pH;
Fig. 4 is a graph representing the optical density (OD) at 230 nm of the
formulation as a function of time during the concentration process as measured
by a spectrophotometer.
Referring to Fig. 1, a process for preparing a ready-to-use aqueous
pharmaceutical formulation comprising a high molecular weight hyaluronic acid
salt at a specified pharmaceutical concentration is shown. Prior to preparing the
formulation, the process plant equipment is cleaned with purified water (step 1),
sterilized with clean steam (step 2), and washed with sterile distilled water for
injection (WFI) (step 3).
A preparation of an aqueous HA formulation, for example a diluted sodium
hyaluronate solution at a concentration less than the concentration specified for
the final pharmaceutical formulation is introduced into a mixing reactor for
preparing the prefiltered formulation (step 4).
The aqueous HA solution may either be prepared from a dried HA salt or from
the solution prepared according to PCT application WO 00/44925, before the
The addition of a concentrated salt solution (25x), dosed by a peristaltic pump,
is carried out in order to add the right amount of salts coupled with the amount
of HA added (step 5). The salt solution normally contains NaCI, buffers and
other excipients specified for the final pharmaceutical formulation, and they are
added in order to bring the pH to a physiological pH, such as 7.4, and to give
the final formulation the physiological osmolarity, such as 300 mOsm/lt.
The salt solution, buffers and other excipients may also be added aseptically
after the filtration step 7 directly into the reaction chamber. This is particularly
advantageous for very high molecular weight HA in view of the fact that salts
decrease the filterability of HA and thus would require greater dilution of the
pre-filtered solution. The amount of excipients added can be monitored in real
time by an electrical conductivity sensor (probe) in the reaction chamber. The
conductivity of the HA formulation is related to the amount of excipient in the
formulation and can be determined empirically. Thus, by measuring the
conductivity of the HA formulation as excipient is added in the reaction chamber
until the required concentration is reached, the required amount of excipient in
the formulation can be added in a simple, reliable and precise manner. The
aforegoing in particular obviates the need to calculate and measure dosages in
advance, and in particular removes the problem of having to take into account
and compensate for the certain amount of HA, even if small, retained by the
filter during the filtration step 7.
By way of example, the formulation may be prepared from 45 grams of dried
sodium hyaluronate of an average molecular weight of 2.2 million Daltons
mixed-with 15 liters of WFI. In this case, due to the high molecular weight of
HA, the salts and buffer solution is added after filtration in order to avoid a
greater dilution of the pre-filtered solution than needed, as mentioned above.
A stirring machine in the reactor mixes the prefiltered solution (step 6), for
example for about 120 minutes, until it is homogeneous. In this particular
example, the prefiltered solution has an HA concentration of 0.3% wt/v. At this
concentration, hyaluronic acid with a molecular weight of 2.2 million Daltons, at
room temperature and physiological pH, has a low viscosity which enables it to
pass entirely through a filter with pore size of 0.22 p. The maximum viscosity
at which all of the HA formulation passes through a filter having a pore size of
0.22 urn, is found to be approximately 5 Pas as measured at 0.1 s-1 shear rate
at 20° C . This maximum viscosity however will depend on the filter pore size: a
smaller pore size, such as 0,1 urn, would lower this maximum viscosity at which
all HA would pass through the filter.
As discussed above, the maximum concentration of HA in an aqueous solution
at which substantially all the HA will pass through a sterilizing filter, for example
a filter of 0.22 µm, will depend on the molecular weight of the hyaluronic acid.
This may be shown with reference to the graphs shown in Figures 2 and 3,
whereby Fig. 2 shows the percentage of sodium hyaluronate at average
molecular weight of about 1.1 million Daltons passing through a filter of 0.22 µm
pore size at substantially ambient temperature (around 20° C) and substantially
neutral pH (around 7) as a function of the concentration (grams of sodium
hyaluronate per deciliter of water - % wt/v).
Fig. 3 shows a similar graph under the same conditions except that the sodium
hyaluronate has an average molecular weight of about 2.2 million Daltons. It
may be seen on the graph of Fig. 2 that all the sodium hyaluronate at a
molecular weight of 1.1 million Daltons passes through the filter up to a
concentration of about 0.92% wt/v (or g/dl), whereas for sodium hyaluronate
having a molecular weight of 2:2 million Daltons, the maximum concentration is
approximately 0.32% wt/v. Therefore, as the molecular weight of the hyaluronic
acid salt increases from 1.1 to 2.2 million Daltons, the maximum concentration
of HA in an aqueous solution to pass 100% through the 0.22 µm filter will
decrease from about 0.94% wt/v to about 0.32% wt/v.
In an industrial process, and considering that after filtration the solution is
concentrated, it is preferred to have a concentration somewhat lower than the
upper limit to ensure that the filtering process is complete and reliable with a
certain margin for error or variations from batch to batch in the molecular weight
of the sodium hyaluronate, the temperature, and the mixing concentrations.
The solution may be forced through the sterilizing filter (step 7) by introducing a
gas under pressure, such a nitrogen, for example at around 3 bars pressure in
the mixing reactor, or by means of a pump. The sterilizing filter preferably has a
pore size of 0.22 µm, but filters having other pore sizes less than 0,45 µm and
greater than around 0.1 µm, may also be used to the extent such filters are or
become commercially available.
When all the solution has passed through the filter into a distiller, the distiller is
sealed off from the filter and mixing reactor with a valve, and a vacuum pump is
activated and regulates the pressure in the distiller by means of a regulatory
valve (steps 8, 9, 10). The pressure is less than 200 millibars in order to bring
the boiling temperature below 60° C, but preferably the pressure is in the region
of 30 to 60 millibars, for example at 40 millibars, whereby the boiling
temperature of water is in the region of 26 to 28° C, close to ambient
Instead of the distiller, a thin film evaporator or any other under-vacuum
concentrators can be useful for a batch, fed-batch or continuous concentration
of the HA formulation.
A heating jacket around the distiller supplies heat energy during the boiling
process. Advantageously, the boiling temperature of less than 30° C ensures
that there is essentially no degradation of the hyaluronic acid such that the
molecular weight of the hyaluronic acid is not reduced.
An HA concentration sensor, advantageously in the form of a
spectrophotometer having an optical fibre immersed in the formulation, may be
provided to measure in real time the concentration of hyaluronic acid (step 11)
and automatically stops the boiling process when the specified concentration is
reached. The spectrophotometer may for example be based on the absorption
of a beam of ultraviolet light (e.g. wave length 230 nm) positioned within the
solution in the distiller. Fig. 4 shows the optical density at 230 nm measured
over time during the whole concentration process (for example up to 2 % w/v.)
The real time HA concentration measurement obviates the need to mix very
precise quantities during the preparation of the diluted prefiltered solution, thus
simplifying the process.
The vacuum boiling also has an important advantage of degassing the
formulation which, in view of the mixing process and application of nitrogen at
high pressure during the filtering process, comprises bubbles, microbubbles
and dissolved gas that are not acceptable in the final pharmaceutical
When the HA concentration sensor in the distiller signals that the specified
concentration is attained, the pressure in the distiller is rapidly increased by
introducing gas, for example nitrogen, therein (step 13) and the jacket heating
around the distiller is cooled to ambient temperature or less, thus immediately
stopping the concentration process of the formulation (step 12).
It may be noted that with a process according to this invention, the
concentration of hyaluronic acid in the formulation may be in the range up to
3%, depending on the specified pharmaceutical use.
The formulation can then be pumped or pushed by a gas (e.g. nitrogen) under
pressure into sterile tanks (steps 28, 30) for filling at another location and/or in
a subsequent stage directly into sterile recipients, such as syringes (step 16),
ready for use. The pharmaceutical formulation may also be directly filled into
sterile recipients for pharmaceutical use without intermediate storage in a sterile
1. A process for preparing a sterile ready-to-use aqueous pharmaceutical
formulation comprising a high molecular weight hyaluronic acid salt (HA)
at a specified concentration, comprising the steps of:
- providing an aqueous formulation comprising high molecular weight HA
at a concentration of less than the specified concentration;
- passing said aqueous formulation through a filter having a pore size not
exceeding 0.45 µm;
- concentrating said aqueous formulation by applying a vacuum and
boiling off water until said specified concentration is reached.
2. Process as claimed in claim 1, wherein after the concentrating step, the
pharmaceutical formulation is filled in sterile recipients ready for use, or
emptied into sterile tanks and subsequently filled in sterile recipients
ready for use.
3. Process as claimed in claim 1 or 2, wherein the vacuum applied in the
concentrating step is at an absolute pressure not exceeding 200
4. Process as claimed in the preceding claim, wherein the vacuum is at a
pressure in the range of 30 to 60 millibars.
5. Process as claimed in any of the preceding claims, wherein the average
molecular weight of HA is in the range of 800'000 to 5'000'000 Daltons.
6. Process as claimed in any one of the preceding claims, wherein the filter
has a pore size not exceeding 0.22 µm.
7. Process as claimed in > any one of the preceding claims, wherein, during
the concentration step, the concentration of HA is measured in real time and
the vacuum boiling process is stopped automatically when the specified
concentration is measured.
8. Process as claimed in any of the preceding claims, wherein the HA
concentration is measured with a spectrophotometer sensing wave radiation
such as herein described) absorption in the formulation.
9. Process as claimed in any one of the preceding claims, wherein
excipients are added to the formulation after the filtration step, and wherein the
conductivity of the HA formulation is measured in real time until the amount of
excipients reaches the required value.
A process for preparing a sterile
ready-to-use aqueous pharmaceutical formulation
comprises a high molecular weight hyaluronic
acid salt (HA) at a specified concentration,
comprising the steps of: providing an aqueous
formulation comprising high molecular weight
HAat a concentration of less than the specified final
concentration; passing said aqueous formulation
through a filter having a pore sizeless than 0.45 pm;
concentrating said aqueous formulation by applying
a vacuum and boiling off water until said specified
concentration is reached.
|Indian Patent Application Number||112/KOLNP/2005|
|PG Journal Number||05/2009|
|Date of Filing||01-Feb-2005|
|Name of Patentee||LABORATOIRE MEDIDOM S. A.|
|Applicant Address||24, AVENUE DE CHAMPEL, CH-1206 GENEVA|
|PCT International Classification Number||A61K 31/728|
|PCT International Application Number||PCT/IB2003/003524|
|PCT International Filing date||2003-08-04|