Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED PROCESS FOR THE MANUFACTURE OF COLLAGEN
Field of the invention
This invention relates to collagen, particularly to an improved form of
collagen
and to a process for preparing improved collagen.
Background Art
Collagen is the major protein component of bone, cartilage, ligament, tendon,
skin and connective tissue in animals. The word 'collagen' is often used as a
generic
term to cover a wide range of protein molecules which share a common triple
helical
structure. However, the most common types of collagen present in connective
tissue
~ o are Types I-III. Type I collagen is the major collagen of skin, tendon and
ligament,
whereas Type III collagen is important in blood vessels. The collagen molecule
is
composed of three collagen polypeptides which form a tight triple helix. The
majority
of each polypeptide is characterised by repeating amino acid sequence Gly-X-Y-
, where
X and Y can be any amino acid but are most usually proline and hydroxyproline.
At
~ s each end of the collagen polypeptide there is a non triple helical
telopeptide region.
The telopeptide regions of the collagen chains are responsible for the
crosslinking
between the chains.
Collagen is synthesised within the cell as precursor collagen (a) chains.
During
further cellular processing the telopeptide regions at each end of the
collagen molecule
zo are formed. These regions play an essential role in the maturation of the
collagen
matrix by intramolecular crosslinking formation which is a crucial step in the
formation
of strong collagenous tissues. In the extracellular matrix, the collagen is
incorporated
into fibrils, which then further associate in tissue to form fibre bundles.
Because of its vital role in all connective tissues, collagen has increasingly
zs become the basis of biomaterials, including native, unmodified tissue
grafts,
manufactured medical products including replacement components for the
cardiovascular system, as well as injectable collagen for soft tissue
augmentation. It is
particularly in the form of injectable or soluble collagen where much recent
research
has been conducted, as such soluble collagen has many medical as well as
cosmetic
ao applications. A range of commercially available collagenous biomaterials
and soluble
s collagen implants are available including ZYDERM and ZYPLAST (injectable
collagen
implants, Collagen Corporation, Palo Alto), ATELOCOLLAGEN (injectable collagen
implants, Koken Company, Tokyo), GELFOAM (gelatin haemostat foams, Upjohn
Co., Michigan) and COLLASTAT (collagen haemostat sponges, Kendall Co.,
Boston).
35 ZYDERM collagen implant (sometimes termed ZCI) is a sterile suspension of
bovine collagen fibrils in 20mM sodium phosphate, 130mM NaCI and 0.3 %
Lignocaine, pH7.2. It is prepared from pepsin solublised collagen which is
precipitated
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2
as f brils which are then collected and resuspended in final buffer to protein
concentrations of either 35 or 65mg/ml. It is then packed into syringes for
delivery.
Electron microscopy reveals that ZCI comprises a polydisperse mixture of
collagen
fibrils. Due to its fibrillar nature, 'its flow behaviour during extrusion
from a syringe is
s important and studies have shown it exhibits non-Newtonian shear thinning
behaviour.
ZYPLAST, which is a more recent product is also an injectable sterile
suspension
of bovine collagen fibrils in phosphate-buffered physiological saline but has
collagen
fibrils stabilised by a low level of glutaraldehyde. The crosslinked collagen
has protein
concentration of 35mg/ml, with a pH of 7-7.6.
~ o Given that soluble collagen, such as in the form of ZYPLAST and ZYDERM is
being injected into skin and other tissues, the primary requirement of all
forms of
soluble collagen is that they are immunologically inert and non toxic. Despite
the fact
that collagen is natural and essential component of living systems, there have
been
reports that soluble collagen as well as collagen based biomaterials, may
induce
~ s cytotoxic effects. One of the main reasons is due to the fact that some
commercial
collagen products provide crosslinked collagen which is very stable. However
crosslinking agents which are used include glutaraldehyde and hyaluronic acid
which
are potential causes of toxicity. It has also been reported that non-
crosslinked soluble
collagen products can be immunogenic, as it has been shown that antibodies can
be
2o generated against the collagen. Studies have indicated that Type I collagen
is an
extremely poor immunogen and is much less likely that other collagen types
such as
Types III, V and VI to lead to an antigenic response. Accordingly, it is
desirable that a
substantially 100% Type I soluble collagen product be available.
Further, the immunogenicity of various commercial injectable soluble collagen
Zs products has been examined, with it being found that very small quantities
of non
collagenous proteins were present which lead to a dominant immunological
response.
Another potential source of immunogenicity was the presence of denatured
collagen, as
individual chains tend to be more immunogenic than intact helical collagen
molecules.
Tests which have been performed to evaluate the general toxicity of collagen
so biomaterials including soluble collagen have been performed on animals
including rats,
guinea pigs and rabbits in order to assess short and long term acute or
chronic
inflammation, as well as immunological testing. Tests have also been performed
on
humans in respect of immunological responses to dermal ZYDERM and ZYPLAST
implants from patients undergoing predominantly aesthetic cosmetic surgery for
ss correction of scars or wrinkles. These products are perhaps the most widely
characterised and used examples of purified and reconstituted collagen explant
material.
On the whole immune response has been low, although in a small number of
patients,
an immunological reaction may be observed.
... ..._... _ _ ~ i
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3
The most common response reported is one of hypersensitivity and around 3 % of
the population will have an underlying reaction. While pre-treatment skin
tests are
routinely performed to assess sensitivity of prospective patients, these do
not appear to
' screen out some patients who will develop an immune response after
injection.
s Accordingly, there is a need for a non-immunogenic injectable soluble
collagen
product.
Further, due to the viscosity of the commercially available injectable soluble
collagen products, injectability through a fine gauge needle (such as 25-30)
is difficult
and blobbing or lumping of the injectate occurs. This is potentially
problematic
~ o particularly where injecting into fme facial lines is required. Further,
often the needle
causes final shearing of the product. Also, constant flow cannot be assured
and often
delivery of the exact amount cannot be achieved. An easily injectable soluble
collagen
product which still has similar collagen concentration is therefore desirable.
The viscosities of currently commercially available collagen products are such
v 5 that when such products are taken out of the lowered temperatures at which
they are
stored, prior to use, they are incapable of being immediately injected. The
collagen
products are so viscous that warming of the product (such as by immersing the
syringe
in warm water) is required. This warming can sometimes cause denaturing of the
collagen. A collagen product having a viscosity such that it is immediately
injectable
zo upon removal from lowered temperatures would therefore be an advantage.
It has also been commonly observed in respect of the commercially available
injectable collagen products that their persistence is variable, which
manifests in
shrinkage and spreading through the dermis of the collagen implant after 6
months, thus
leading to patients requiring a supplementary injection of implant collagen. A
z5 persistent collagen implant retaining constant volume and having an
enhanced resistance
to degradation is accordingly required.
Further, the commercial collagen products currently available on the market
are a
"white to off white" colour. It has therefore been observed that when patients
with
darker skin colour (Caucasians as well as non Caucasians) are administered a
soluble
so collagen injection into the skin, the collagen implant is quite often
discernible under the
skin as a whitish area. This is particularly the case where injection has
occurred fairly
superficially such as to treat fine facial lines. This of course is extremely
undesirable,
given that usually the injection has been undertaken for cosmetic reasons.
Objects of the invention
35 Accordingly, it is an object of this invention to provide a collagen
composition
which is translucent to clear in appearance, which is of high purity, which is
immunologically inert, which is of such viscosity that it can be easily
injected at
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4
temperatures in the range of from 8-18°C and which after injection into
the skin, lasts
without dissipating or disappearing fox a period of 9-24 months.
It is another object of this invention to provide a process for the
preparation of an
improved collagen composition which is translucent to clear in appearance,
which is of
s high purity, which is of such viscosity that it can be easily injected at
temperatures in
the range of from 8-18°C and which after injection into the skin, lasts
without
dissipating or disappearing for a period of 9-24 months.
It is another object of the present invention to provide a collagen
composition
made by the above process.
~ o It is a further object of the present invention to provide a syringe
prefilled with an
improved collagen composition which is translucent to clear in appearance,
which is of
high purity, which is of such viscosity that it can be easily injected at
temperatures in
the range of from 8-18°C and which after injection into the skin, lasts
without
dissipating or disappearing for a period of 9-24 months.
i s It is a further object of the present invention to provide a method of
soft tissue
augmentation which comprises administering an improved collagen composition of
the
present invention, to a patient in need thereof.
Statement of Invention
A first aspect of this invention provides an injectable collagen composition
Zo comprising substantially 100% Type I collagen, said injectable composition
having a
pH in the range of from about 3 to 6 and a collagen concentration of from
about 20-90
mglml .
According to an embodiment of the first aspect of this invention, there is
provided
an injectable collagen composition comprising substantially 100% Type I
collagen, said
zs injectable composition having a pH in the range of from about 3 to 6 and a
collagen
concentration of from about 20-90 mg/ml and a viscosity such that said
composition is
injectable through at least a 30 gauge needle without blobbing.
According to a further embodiment of the first aspect of the present
invention,
there is provided an injectable collagen composition comprising substantially
100%
so Type I collagen, said injectable composition having a pH in the range of
from about 3
to 6 and a collagen concentration of from about 20-90 mglml, and a viscosity
such that
said composition is injectable through at least a 30 gauge needle without
blobbing at a
temperature in the range of from about 8 to 18°C.
According to a further embodiment of the first aspect of the present
invention,
s~ there is provided an injectable collagen composition comprising
substantially 100%
Type I collagen, said injectable composition having a pH in the range of from
about 3
_.. . _........._ _. r i ,
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to 6 and a collagen concentration of from about 20-90 mglml, and wherein said
composition is substantially translucent to clear in appearance.
Typically, the pH of the composition is in the range of from about 3.5-5.5,
more
typically in the range of from about 4-5 and most typically in the range of
from about
5 4.2-4.8.
Typically, the collagen concentration is in the range of from about 20-60
mg/ml,
more typically in the range of from about 20-40mg1m1, most typically in the
range of
from about 22-38mg1m1.
Typically the injectable composition is injectable using a 30-35 gauge needle,
~ o most typically a 32 or 33 gauge needle is used.
Typically, the viscosity of the composition is such that it is injectable
through a
30-35 gauge needle when the composition is at a temperature within the range
of 10-15°
C, more typically a temperature in the range of from 10-13°C .
According to a second aspect of the present invention, there is provided a
process
5 for the preparation of a collagen composition, said process comprising the
steps of:
(a) mechanically processing mammalian connective tissue to form connective
tissue fragments of from about 0.1-Smm in length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme,
20 (c) mechanically shearing said solution such that substantially all
collagen in the
solution is capable of passing through a 200 micron filter,
(d) adding a non-phosphate salt andlor a non-phosphate buffer to said sheared
solution to form a collagen suspension; and
(e) dialysing the collagen suspension to a collagen concentration of from
about 20-
25 90mg1m1.
Typically, the mehanical processing includes any mechanical means for breaking
up or fragmenting the tendons. Usually, such mechanical processing can be any
form
of comminuting such as dicing, chopping, homogenising or other physical
treatment
and most typically takes the form of mincing. The minced tendon fragments
which
so result are more typically in the size range of from about 0.5-4mm, most
typically 1-
3mm.
In an embodiment of the second aspect of the present invention there is
provided a
process for the preparation of a collagen composition, said process comprising
the steps
of:
35 (a) mechanically processing mammalian connective tissue to form connective
tissue fragments of from about 0.1-Smm in length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme,
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(c) mixing said solution,
(d) mechanically shearing said solution such that substantially all collagen
in the
solution is capable of passing through a 200 micron filter,
(e) adding a non-phosphate salt and/or a non-phosphate buffer to said sheared
s solution to form a collagen suspension; and
(f) dialysing the collagen suspension to a collagen concentration of from
about 20-
90mg/ml .
Typically the mixing of the solution occurs in a range of 3500-22000rpm, such
that polypeptide chains of each collagen molecule in said solution at least
begin to
~ o unweave from each other.
Typically, the mammalian connective tissue can be obtained from porcine and
bovine origin and most typically includes skin, cartilage, tendon and muscle.
Accordingly, in an embodiment of the second aspect of the present invention,
there is provided a a process for the preparation of a collagen composition,
said process
i s comprising the steps of:
(a) mechanically processing bovine tendons to form bovine tendon fragments of
from about 0.1-Smm in length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme,
20 (c) mixing said solution,
(d) mechanically shearing said solution such that substantially all collagen
in the
solution is capable of passing through a 200 micron filter,
(e) adding a non-phosphate salt andlor a non-phosphate buffer to said sheared
solution to form a collagen suspension; and
z5 (f) dialysing the collagen suspension to a collagen concentration of from
about 20-
90mg/ml.
Typically, the bovine tendons are obtained from young bovines, most typically
bovines which are up to about 1 year in age and which are termed 'vealers',
the bovine
tendons most preferred in this process effectively being veal tendons.
so Typically the connective tissue fragments have a diameter similar to their
length.
Accordingly, it is preferable that the connective tissue fragments have both
length and
diameter in the range of from about 0.1-Smm.
In one embodiment of the second aspect of the present invention, there is
provided a process for the preparation of a collagen composition, said process
s5 comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Smm in length and diameter,
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(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme,
(c) mixing said solution,
(d) mechanically shearing said solution such that substantially all collagen
in the
s solution is capable of passing through a 200 micron filter,
- (e) adding a non-phosphate salt and/or a non-phosphate buffer to said
sheared
solution to form a collagen suspension; and
(f) dialysing the collagen suspension to a collagen concentration of from
about 20-
90mg/ml.
~ o Typically, the sterile conditions include treating the bovine tendons with
bleach or
some other sterilizing or antiseptic agent to ensure a pyrogen free
environment. Such
treatment may typically be the chemical sterilisation of the tendons in dilute
hypochlorite solution. Alternatively, it is also typical that the bovine
tendons are first
physically processed to fragments which are then chemically sterilised. Most
typically,
i 5 immersion of the tendons in dilute (250ppm) sodium hypochlorite solution
for any time
from about several minutes to about two hours occurs.
After sterilisation, the tendons or fragments are typically washed to remove
the
sterilisation agent.
In another embodiment of the second aspect of the present invention, there is
zo provided a process for the preparation of a collagen composition, said
process
comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Smm in length,
(b} digesting said fragments in acidic solution containing at least one
proteolytic
zs (non collagenase) enzyme at a temperature range of from about 4-
25°C,
(c) mixing said solution,
(d) mechanically shearing said solution such that substantially all collagen
in the
solution is capable of passing through a 200 micron filter,
(e) adding a non-phosphate salt and/or a non-phosphate buffer to said sheared
so solution to form a collagen suspension; and
(f) dialysing the collagen suspension to a collagen concentration of from
about
20-90mg1m1.
' Typically, the acidic solution in which digestion occurs is selected from
any
carboxylic acid solution or from any mineral acid solution or a mixture of
both. More
ss typically the acidic solution is acetic acid solution which may or may not
be mixed with
fuming concentrated HCI. The pH of the digestion solution is in the range of
from
about 1 to about 6.
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It is also typical that the enzyme present in the acidic solution is selected
from
pepsin, trypsin and any other non-collagenase enzyme. Most typically, the
enzyme is
pepsin.
Usually the digestion occurs at a temperature of between 8-20°C, more
usually
s 8-15°C and most typically 8-12°C.
It is also usual that some mixing of the digestion solution occurs either
prior to
just after digestion begins.
The time for digestion is variable, but typically occurs in the range of from
about
4 to 28 hours, more typically 5-24 hours, most typically in the range of from
about 5-
~ 0 20 hours.
Most typically the digestion solution comprises for each 100g of tendon
fragments, 30 litres of 10% acetic acid, 20m1 fuming HCl and lg of pepsin.
In another embodiment of the second aspect of the present invention, there is
provided a process for the preparation of a collagen composition, said process
i s comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-5mm in length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
2o period of from about 4-28 hours,
(c) mixing said solution,
(d) mechanically shearing said solution such that substantially all collagen
in the
solution is capable of passing through a 200 micron filter,
(e) adding a non-phosphate salt and/or a non-phosphate buffer to said sheared
zs solution to form a collagen suspension; and
(f) dialysing the collagen suspension to a collagen concentration of from
about 20-
90mg/ml.
Typically, the digesting occurs for up to 24 hours at 8-10°C.
The solution is then submitted to mixing, preferably in the form of mechanical
ao blending, such as a laboratory blender.
Optionally the solution may be treated in order to inactivate the enzyme
and/or to
remove traces of the acid and enzyme. Such treatment, can occur either before
or after
the mixing of the digestion solution which is undertaken to unweave the
collagen
fragments .
35 Typically, the blended solution is chilled to a temperature of about 5-
10°C for a
period of up to about 24 hours before it is mechanically sheared.
Typically, the mechanical shearing of the solution takes the form of
filtering.
Sieving is also another means by which shearing of the collagen in solution
can occur.
,.
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Thus, in another embodiment of the second aspect of the present invention,
there
is provided a process for the preparation of a collagen composition, said
process
comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
s bovine tendon fragments of from about 0.1-Smm,
' (b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
period of from about 4-28 hours,
(c) blending said solution at a speed of from about 3500-22,OOOrpm,
~ o {d) filtering said solution such that after filtering said solution
contains
substantially all collagen molecules in a size range of from about 50-200
microns,
(e) adding a non-phosphate salt and/or a non-phosphate buffer to said filtered
solution to form a collagen suspension; and
(f) dialysing the collagen suspension to a collagen concentration of from
about 20-
i s 90mglml.
Filtering of the blended solution occurs through the use of 50-100 micron
filters,
most typically a 50 micron filter is used. The filtering results in
substantially all the
collagen molecules being sheared to a size of about 50-200 microns, most
preferably 50
microns .
Zo Typically, the filtered solution containing the collagen molecules is then
treated
with an alkali metal non-phosphate salt solution sufficient to reconstitute
the collagen
from the solution. The reconstituted collagen appears as a collagen suspension
and is
caused by the collagen in solution reaggregating into fibrils Typically such
alkali metal
salts include sodium hydroxide, sodium chloride and potassium chloride.
is Typically, the filtered solution which contains the collagen molecules in
solution
is then treated with sodium chloride, preferably in the form of sodium
chloride
solution, most preferably in the form of 5M sodium chloride solution.
Typically
sufficient sodium chloride is added to the filtered solution such that the
filtered solution
is saturated and the collagen molecules come out of solution. More typically,
sufficient
so sodium chloride is added to the filtered solution such that a 1M
concentration of sodium
chloride is obtained in the final solution which results in reconstitution of
the collagen
molecules.
Thus, in another embodiment of the second aspect of the present invention,
there
is provided a process for the preparation of a collagen composition, said
process
~ 3s comprising the steps of:
{a} mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Smm in length and diameter,
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(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
period of from about 4-28 hours,
(c) blending said solution at a speed of from about 3500-22,OOOrpm,
s (d) filtering said solution through at least one filter having a filter size
range of
from about 50-200 microns,
(e) adding sodium chloride to said filtered solution such that a collagen
suspension
forms; and
(f) dialysing the collagen suspension to a collagen concentration of from
about 20-
i o 90mg1m1.
Typically the sodium chloride is added to the filtered solution with stirring.
It is also usual that the solution containing the sodium chloride is left to
rest for a
period of from about 1-24 hours, more usually 1-15 hours. Typically the
temperature
at which the resting occurs is in a range of from about 8-25°C, more
typically from
about 8-15°C.
Typically, the reconstituted collagen molecules in the form of a collagen
suspension in the solution are collected by gentle batch centrifugation, the
supernatant
being discarded. The centrifugation will also serve to remove any remaining
enzyme,
particularly if the solution has not been treated since digestion. Usually,
the collagen
zo gel can be resuspended in buffer or water and centrifuged again, with the
supernatant
again being discarded.
Thus, in another embodiment of the second aspect of the present invention,
there
is provided a process for the preparation of a collagen composition, said
process
comprising the steps of:
is (a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Smm in both diameter and length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
period of from about 4-28 hours,
ao (c) blending said solution at a speed of from about 3500-22,OOOrpm,
(d) filtering said solution through at least one filter having a filter size
range of
from about 50-200 microns,
(e) i) adding sodium chloride to said filtered solution to form a collagen
suspension;
ss ii) centrifuging the solution in order to provide collagen gel; and
(~ dialysing the collagen gel to a collagen concentration of from about 20-
90mg1m1.
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In another embodiment of the second aspect of the present invention, there
is provided a process for the preparation of a collagen composition, said
process
comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Srnm in both diameter and length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
period of from about 4-28 hours,
(c) blending said solution at a speed of from about 3500-22,000rpm,
i o (d) filtering said solution through at least one filter having a filter
size range of
from about 50-200 microns,
(e) i) adding sodium chloride to said filtered solution to form a collagen
suspension;
ii) centrifuging the solution in order to provide collagen gel,
~ s (f) collecting and resuspending the collagen gel and again centrifuging
said
resuspended collagen to provide collagen gel; and
(g) dialysing the collagen gel to a collagen concentration of from about 20-
90mg/ml.
Typically the dialysis in undertaken using dialysis bags or tubes. Usually,
the
2o collagen is dialysed against distilled water or dilute acid such as acetic
acid. Preferably
the dialysis occurs over a period of from about 12-96 hours, more preferably
over a
period of from about 24-80 hours, most preferably over a period of from 48-72
hours.
Typically over the dialysis period, the collagen is dialysed against between 1
to 6
changes of water or other dialysate, more typically between 1 to 4 changes of
water,
2s most typically against 3 changes of water. In a preferred embodiment, the
dialysis of
the collagen occurs against 3 changes of water at 24 hour intervals.
The concentration of the collagen in the dialysis tubes or bags should
typically
reach the required range of from about 20-90 mglml after dialysis for a period
of about
24-72 hours. More typically, the concentration is in the range of from 25-
60mg1m1,
so most typically in the range of from 25-40 mglml.
The viscosity of the collagen solution is typically monitored such that the
final
collagen solution has a viscosity which allows it to be injected without
blobbing through
a syringe having a needle in the range of from about 30-35 gauge. Further, the
viscosity is such that the collagen composition is injectable when at a
temperature in the
ss range of from about 8-18°C, more typically it is injectable when at
a temperature in the
range of from about 10-15°C.
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12
In a further embodiment of the second aspect of the present invention, there
is
provided a process for the preparation of a collagen composition, said process
comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
s bovine tendon fragments of from about 0.1-Smm in length,
(b) digesting said fragments in acidic solution containing at least one
proteolytic
(non collagenase) enzyme at a temperature range of from about 4-25°C
and over a
period of from about 4-28 hours,
(c) blending said solution at a speed of from about 3500-22,OOOrpm,
~ o (d) filtering said solution such that after filtering said solution
contains
substantially all collagen molecules in a size range of from about 50-200
microns,
(e) i) adding sodium chloride to said filtered solution to form a collagen
suspension;
ii) centrifuging the solution in order to provide collagen gel,
~ s (f) dialysing the collagen gel to a collagen concentration of from about
20-
90mg/ml; and
(g) adding an anaesthetic agent to the collagen to give a anaesthetic agent
concentration of from about 2-Smg/ml.
Typically, the anaesthetic agent is Lignocaine solution which is added to the
2o collagen with mixing, such as in the form of gentle centrifugation, in
order that any air
bubbles may be removed prior to use of the collagen solution. More typically,
the final
Lignocaine concentration of the collagen solution is from about 3-5 mg/ml,
most
typically 3-4mg/ml.
In a preferred embodiment of the second aspect of the present invention, there
is
25 provided a process for the preparation of a collagen composition, said
process
comprising the steps of:
(a) mechanically processing bovine tendons under sterile conditions to form
bovine tendon fragments of from about 0.1-Smm in both diameter and length,
(b) digesting said fragments in acidic solution containing pepsin at a
temperature
so range of from about 4-25°C and over a period of about 24 hours,
(c) blending said solution at a speed of from about 3500-22,OOOrpm,
(d) filtering said solution through a 50 micron filter such that after
filtering said
solution contains substantially all collagen molecules of a size of about 50
microns,
(e) i) adding sodium chloride to said filtered solution to form a collagen
s5 suspension;
ii) centrifuging the solution in order to provide collagen gel,
(fj collecting and resuspending the collagen gel and again centrifuging said
resuspended collagen to provide collagen gel,
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(g) dialysing the collagen gel against three changes of distilled water at 24
hour
intervals to a collagen concentration of from about 20-90mg/ml,
(h) adding Lignocaine HCl solution to the collagen to give a Lidocaine
concentration of from about 2-Srng/ml; and
s {i) centrifuging to remove air from the collagen.
A third aspect of the present invention provides an injectable collagen
composition produced by any one of the above processes.
A fourth aspect of the present invention provides a syringe prefilled with an
injectable collagen composition comprising substantially 100% Type I collagen,
said
i o injectable composition having a pH in the range of from about 3 to 6 and a
collagen
concentration of from about 20-90 mg/ml and a viscosity such that said
composition is
injectable through at least a 30 gauge needle without blobbing.
An embodiment of the fourth aspect of the present invention provides a syringe
prefilled with an injectable collagen composition comprising substantially 100
% Type I
~ s collagen, said injectable composition having a pH in the range of from
about 3 to 6 and
a collagen concentration of from about 20-90 mg/ml and a viscosity such that
said
composition is injectable through at least a 30 gauge needle without blobbing
when said
composition is at a temperature in the range of from about 8 to 18°C.
According to a further embodiment of the fourth aspect of the present
invention,
Zo there is provided a syringe prefllled with an injectable collagen
composition comprising
substantially 100% Type I collagen, said injectable composition having a pH in
the
range of from about 3 to 6 and a collagen concentration of from about 20-90
mg/ml and
wherein said composition is substantially translucent to clear in appearance.
Typically, the pH of the composition is in the range of from about 3.5-5.5,
more
25 typically in the range of from about 4-5 and most typically in the range of
from about
4.2-4.8.
Typically, the collagen concentration is in the range of from about 20-60
mglml,
more typically in the range of from about 20-40mg/ml, most typically in the
range of
from about 22-38mg/m1.
3o In a fifth aspect of the present invention there is provided a method of
augmenting
soft tissue which comprises injecting a composition of the present invention
to a patient
in need thereof.
Typically such augmentation includes the repair or correction of defects in
soft
tissue.
~ ss Typically, such defects include frown lines, nasolabial creases, wrinkles
and other
superficial sclerodermal lines .
The product of the invention is translucent to clear and is not visible when
injected into people having dark skin pigment. The product of the invention is
non
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turbid, non-immunogenic, injectable without bIobbing through at least a 30
gauge
needle and is also non crosslinked and of high purity.
The product is also of such viscosity that it is injectable when at a
temperature in
the range of about 8-18°C, and~even when at a temperature of 10-
12°C.
s For the purposes of the present specification, the following terms are
defined
below.
"Collagen" refers to all types and forms of collagen from any source, whether
invivo or invitrc~ produced, and is not limited to cross linked collagen but
includes non-
crosslinked collagen fibrils.
i o "Collagen in solution" refers to collagen which is in an acidic solution
such that
the collagen is in the non-fibrillar form.
"Injectable" refers to compositions that can be dispensed from at least 30
gauge
syringes under normal conditions and manual pressure without substantial
blobbing.
"Clear" refers to the optical clarity wherein the product has substantially at
least
~ s 90% transmittance of light at 410nm.
"Translucent" also refers to the optical clarity wherein the product has
substantially at least 75 % transmittance of light at 410nm.
"Blobbing" refers to the coagulating and/or lumping of the injectate. It also
refers to the inability of a composition to be injected at a constant rate.
zo Best mode and other modes for carrying out the invention
Preparation of Raw Material
The bovine tendons or other mammalian connective tissue are processed in
sterile
conditions. Accordingly, the tissue is washed in bleach such as dilute
hypochlorite
solution as well as washed in sterile distilled water, so that the tissue and
processing
Zs conditions are pyrogen free. Clean room conditions using sterile forceps
and other
instruments and wearing of sterile protective clothing is also observed.
Each tissue is differently processed according to what form of tissue it is.
Typically, if the skin is being utilised as the collagen source, depilation
and removal of
any fat occurs. In the case of tendons, the skin is removed along with any fat
and
so unwanted tissue.
Mechanical processing of the tissue takes the form of comminution by grinding,
mincing or such similar physical treatment so that the tissue is finely
fragmented or
minced which enables collagen solubilisation to occur more effectively.
Solubilisation
35 The collagen in the tissue is solubilised under non-denaturing conditions
by
dispersing the tissue fragments in an aqueous acidic medium and digesting with
a
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proteolytic enzyme other than a collagenase. The pH of the medium can be
anywhere
in the range of from about 1 to less than 7. Mineral acids such as HCl or
carboxylic
acids such as acetic and malonic, or a mixture of both carboxylic and mineral
acids can
' be used to create the acidic medium. An aqueous acidic solution comprising a
mixture
s of acetic acid with a small amount of concentrated fuming HCl is preferred
for the
' purposes of solubilisation. It is also preferable that the temperature at
which
solubilisation occurs is kept in the range of from about 4-25°C in
order to avoid
denaturation.
The enzyme should be active at acidic pH and low temperatures and is usually
selected from such enzymes as pepsin, papain, trypsin and chymotrypsin. The
enzyme
can be present in the acidic solution prior to the addition of the tissue, or
alternatively
can be added to the acidic solution after the tissue. The enzyme concentration
is
typically in the range of from about 0.1 % to 5 % by weight.
Digestion of the tissue with the enzyme can occur over any time from several
15 hours to weeks. At least 4 hours is preferred for digestion at lowered
temperatures in
the order of 5-15°C.
Initial stirring and thereafter occasional stirring is usual during the
solubilisation.
Blending
The solution is submitted to stirring in a blender which is performed in order
to
zo help unweave the collagen fragments. Filtering and reconstitution is
performed more
easily and effectively on at least partly unwound collagen strands. The
blending occurs
at a speed in the range of from about 3500 to 22000 rpm. Conveniently,
blending can
occur at a temperature in the range of from about 15-25 °C, and usually
at around 20°
C.
zs Filtering
Filtering provides a preferred starting material by breaking up any fibrillar
aggregates in the solution. Most typically, the solution which has been
chilled is
mechanically filtered through a 50 micron filter at temperatures, in the order
of 5-25°C.
This results in the collagen having improved flow characteristics and enhanced
so injectability through fme gauge needles.
Reconstitution
Effectively, the aim of reconstitution is to reaggregate collagen fibrils such
that
~ they precipitate out of the filtered collagen solution in the form of a
suspension. This
can be effected by raising the ionic strength and/or pH of the solution by
such means as
ss adding a nonphosphate buffer and/or a non phosphate salt solution (which
may or may
not be buffered, but if buffered, a non-phosphate buffer is used) to a level
where the
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collagen in solution re-aggregates into fibrils . Accordingly, the pH is
preferably in the
range of from about 2-8, and most preferably in the range of from about 3.5 to
about
7.5.
Sterile NaCI, typically in the form of a solution is added in accordance with
the
s present invention with stirring, to the solution. The addition can occur at
temperatures
in the range of from about 4-25°C, usually around 20°C.
Temperatures are preferably
kept low to avoid crosslinking of the collagen fibrils. The addition of the
salt solution
which can be buffered, but with non-phosphate buffers, results in the collagen
in
solution re-aggregating into atelopeptide fibrils.
~ o Fibril formation can take any time from 30 mins through to more than 24
hours.
Centrifugation
The reaggregated collagen fibrils can be collected by centrifugation.
Centrifugation preferably occurs for at least 30 minutes at an approximate
speed of
about 20,000-40,000 g.
i s The precipitated collagen gel can be resuspended in water or other buffer
if
required and the centrifugation process repeated. This acts to remove any
remaining
enzyme and also is a further purification step. The collagen gel is then
transferred to
sterile dialysis tubes or bags.
Dialysis
2o Dialysis in sterile dialysis tubes or bags against three changes of
distilled sterile
water, with each dialysis period lasting for approximately 20-24 hours is
typical. The
dialysis preferably occurs at such temperatures to avoid crosslinking of the
collagen
fibrils. Typically dialysis occurs at temperatures in the range of about 5-
25°C, usually
around 20°C. The dialysis tubes preferably have a molecular weight cut
off in the
is range of about 5,000-10,000. The concentration of the collagen in the
dialysis
tubeslbags can be monitored until the desired concentration is reached. A
variety of
methods are available for the characterisation of soluble collagen, including
gel
electrophoresis (SDS-PAGE), which demonstrates the purity of the collagen and
the
collagen types present, and amino acid analysis which further distinguishes
the collagen
so types present.
Final Injectable Composition
A local anaesthetic agent, preferably Lignocaine, is added to the collagen to
a
concentration of about 0.2-0.5 % by weight to reduce local pain upon
injection. The
collagen is then loaded into syringes having at least a 30 gauge needle,
preferably a 31,
35 32 or 33 gauge needle.
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Example
Equipment and materials
All equipment is sterile and all sterile manipulations are performed in a
Class A
laminar air flow cabinet in a Class C clean room.
s All non-self sterilising process fluids are sterile filtered (0.2p.).
The following equipment and products were used in the performance of the
present invention:
Sterile, pyrogen free water
t o Clean Room Class C
Laminar Air Flow cabinet Class A (LAF)
Clean Room Attire
Cool Room
Scalpels and Blades
15 Plastic Chopping Board
BP Hypochlorite Solution
Mincer (chemically sterilised with Hypochlorite)
Carboys with sterile venting
blaring Blender
2o Disposable 0.2~ capsule filters
50~. polypropylene filter cartridge in stainless steel housing
BP glacial acetic acid
BP Sodium Chloride
USP Pepsin
2s AR fuming hydrochloric acid
Hydrated dialysis tubing (Gamma sterilised)
Sterile funnel
BP lignocaine HCl
Disposable centrifuge bottles
so Sterile syringes with 32 gauge needles
Process
1. Frozen bovine Achilles tendons are supplied.
2. The tendons are thawed and 1 cm of the ends removed with sterile scalpel.
3. The tendons are chemically sterilised in dilute (250 ppm) sodium
35 hypochiorite solution.
4. The tendons are minced and the mince collected in the LAF.
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5. The mince is weighed and transferred to sterile carboys containing ger each
100g of mince, 30 litres of 10% acetic acid, 20 ml of fuming HCl and lg of
Pepsin.
6. The mixture is allowed to digest for 24 hours.
7. The mixture is then blended in the blaring blender in batches and
recombined in a carboy.
8. The solution is chilled for a period of time (can be up to 24 hours) and
then
filtered through a SOp. filter and collected in a carboy.
9. SM Sodium chloride solution is added with stirring to make the
concentration 1M sodium chloride and the solution is chilled.
~ 0 10. The collagen gel which forms is collected by batch centrifugation. The
supernatant is discarded and the collagen resuspended.
11. The collagen is centrifuged as before and the supernatant discarded.
12. The gel is transferred to sterile dialysis tubes of about 30 cm in length
and
dialysed against 3 changes of water at 24 hour intervals.
13. A representative sample of gel is analysed to determine collagen
concentration.
14. The collagen is concentrated in the dialysis bag to above the required
concentration.
15. The collagen gel is collected and the appropriate amount of Iignocaine HCL
2o solution is added to give the concentration of collagen as about 20-90mg/ml
and the
concentration of lignocaine HCI as about 1-Smg/ml.
16. The product is thoroughly mixed, preferably by gentle centrifugation, to
remove any air prior to filling the syringes fitted with 30-35 gauge needles
with the
collagen product.
Analysis of Bovine Collagen Product
Sam le Bovine Colla en
Batch Number
Date
Descri tion Odour Odourless
Descri tion Translucent-clear e1 mass
Identification (SDA-PAGE) 2 major bands, due to collagen
ocl[I] and a
2[I] chains (monomers) are present.
Higher
molecular weight bands, due to
collagen p-
(dimer) and y-chains (trimer)
are also
resent.
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Identification (SDS-PAGE with 2 major bands, due to collagen
al [I] and a
delayed reduction) 2[I] are present. No additional
band due to
a 1 [III] chains (monomers) is
present.
Higher molecular weight bands,
due to
collagen ~3- (dimer) and y-chains
(trimer)
are also resent.
ELISA Type I collagen detected. Types
III and V
not detected.
Identification (Native PAGE) 1 major band, due to monomeric
collagen is
present. No lower molecular weight
bands
are present. Faint, higher molecular
weight
bands, due to dimer and polymer
collagen
are resent.
Transmittance (%T at 430 nm) Forms clear solution as 0.5%
solution in 50
mM acetic acid.
pH (20C, 0.5% in milliQ water) 4.5 .
Arsenic <2 ppm
Hea Metals (as Pb <5 ppm
Amino acid analysis 11.5% (w/w) hydroxyproline
0.45% (w/w) tyrosine
Concentration 2.6% (w/w) by Kjeldahl nitrogen
analysis
2.6% (wlw) by amino acid analysis
P ro en test Rabbit Passes.
Coliform Grou Passes.
Total Bacterial Count Passes.
