AU729068B2 - Improved process for the manufacture of collagen - Google Patents

Description

WO 98/44809 PCT/!AU98!00226 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 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 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 chains. During further cellular processing the telopeptide regions at each end of the collagen molecule 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 become the basis of biomaterials, including native, unmodified tissue grafts, manufactured medical products including replacement components for the cardiovascular systemn, 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 applications. A range of commercially available collagenous biomaterials and soluble 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).

ZYDERM collagen implant (sometimes termed ZCI) is a sterile suspension of bovine collagen fibrils in 20mM sodium phosphate, 130mM NaCl and 0.3% Lignocaine, pH7.2. It is prepared from pepsin solublised collagen which is precipitated WO 98/44809 PCT/AU198!00226 2 as fibrils 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 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.

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 is 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 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 products has been examined, with it being found that very small quantities of noncollagenous 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 molecuIles.

Tests which have been performed to evaluate the general toxicity of collagen 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 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.

WO 98/44809 PCT/AU98!00226 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.

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 particularly where injecting into fine 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 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 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 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 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 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 WO 98/44809 PCT/AU98/00226 4 temperatures in the range of from 8-18 0 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 this invention to provide a process for the preparation of 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 0 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.

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 0 C and which after injection into the skin, lasts without dissipating or disappearing for a period of 9-24 months.

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 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.

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 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.

UAccording to a further embodiment of the first aspect of the present invention, 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 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 at a temperature in the range of from about 8 to 18 0

C.

According to a further embodiment of the first aspect of the present invention, 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 WO 98/44809 PCT/AU98/00226 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 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 mg/ml, more typically in the range of from about 20-40mg/ml, most typically in the range of from about 22-38mg/ml.

Typically the injectable composition is injectable using a 30-35 gauge needle, 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 0

C.

According to a second aspect of the present invention, there is provided a process for the preparation of a collagen composition, said process comprising the steps of: mechanically processing mammalian connective tissue to form connective tissue fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 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 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: mechanically processing mammalian connective tissue to form connective tissue fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, WO 98/44809 PCT/AU98/00226 6 mixing said solution, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 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 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 comprising the steps of: mechanically processing bovine tendons to form bovine tendon fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, mixing said solution, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 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.

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 0.1-5mm.

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 comprising the steps of: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in length and diameter, WO 98/44809 PCT/AU98/00226 7 digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, mixing said solution, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 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, 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 provided a process for the preparation of a collagen composition, said process comprising the steps of: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0

C,

mixing said solution, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 20-90mg/ml.

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 typically the acidic solution is acetic acid solution which may or may not be mixed with fuming concentrated HCL. The pH of the digestion solution is in the range of from about 1 to about 6.

WO 98/44809 PCT/AU98/00226 8 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 0 C, more usually 8-15 0 C and most typically 8-12 0

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 20 hours.

Most typically the digestion solution comprises for each 100oog of tendon fragments, 30 litres of 10% acetic acid, 20ml fuming HCI and Ig 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 comprising the steps of: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, mixing said solution, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about Typically, the digesting occurs for up to 24 hours at 8-10 0

C.

The solution is then submitted to mixing, preferably in the form of mechanical 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.

Typically, the blended solution is chilled to a temperature of about 5-10 0 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.

WO 98/44809 PCT/AU98/00226 9 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: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, blending said solution at a speed of from about 3500-22,000rpm, filtering said solution such that after filtering said solution contains substantially all collagen molecules in a size range of from about 50-200 microns, adding a non-phosphate salt and/or a non-phosphate buffer to said filtered solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 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 microns.

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.

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 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 comprising the steps of: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in length and diameter, WO 98/44809 PCT/AU98/00226 digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, blending said solution at a speed of from about 3500-22,000rpm, filtering said solution through at least one filter having a filter size range of from about 50-200 microns, adding sodium chloride to said filtered solution such that a collagen suspension forms; and dialysing the collagen suspension to a collagen concentration of from about 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 0 C, more typically from is about 8-15 0

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 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: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in both diameter and length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, blending said solution at a speed of from about 3500-22,000rpm, filtering said solution through at least one filter having a filter size range of from about 50-200 microns, i) adding sodium chloride to said filtered solution to form a collagen suspension; ii) centrifuging the solution in order to provide collagen gel; and dialysing the collagen gel to a collagen concentration of from about WO 98/44809 PCT/AU98/00226 11 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: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in both diameter and length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, blending said solution at a speed of from about 3500-22,000rpm, filtering said solution through at least one filter having a filter size range of from about 50-200 microns, i) adding sodium chloride to said filtered solution to form a collagen suspension; ii) centrifuging the solution in order to provide collagen gel, collecting and resuspending the collagen gel and again centrifuging said resuspended collagen to provide collagen gel; and dialysing the collagen gel to a collagen concentration of from about Typically the dialysis in undertaken using dialysis bags or tubes. Usually, the 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, 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 mg/ml after dialysis for a period of about 24-72 hours. More typically, the concentration is in the range of from 25-60mg/ml, most typically in the range of from 25-40 mg/ml.

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 range of from about 8-18 0 C, more typically it is injectable when at a temperature in the range of from about 10-15 0

C.

WO98/44809 PCT/AU98/00226 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: mechanically processing bovine tendons under sterile conditions to form s bovine tendon fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme at a temperature range of from about 4-25 0 C and over a period of from about 4-28 hours, blending said solution at a speed of from about 3500-22,000rpm, filtering said solution such that after filtering said solution contains substantially all collagen molecules in a size range of from about 50-200 microns, i) adding sodium chloride to said filtered solution to form a collagen suspension; ii) centrifuging the solution in order to provide collagen gel, dialysing the collagen gel to a collagen concentration of from about and adding an anaesthetic agent to the collagen to give a anaesthetic agent concentration of from about Typically, the anaesthetic agent is Lignocaine solution which is added to the 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/mnl, most typically 3-4mg/ml.

In a preferred 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: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in both diameter and length, digesting said fragments in acidic solution containing pepsin at a temperature range of from about 4-25 0 C and over a period of about 24 hours, blending said solution at a speed of from about 3500-22,000rpm, 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, i) adding sodium chloride to said filtered solution to form a collagen suspension; ii) centrifuging the solution in order to provide collagen gel, collecting and resuspending the collagen gel and again centrifuging said resuspended collagen to provide collagen gel, WO 98/44809 PCT/AU98/00226 13 dialysing the collagen gel against three changes of distilled water at 24 hour intervals to a collagen concentration of from about 20-90mg/ml, adding Lignocaine HCI solution to the collagen to give a Lidocaine concentration of from about 2-5mg/ml; and 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 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 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 0

C.

According to a further embodiment of the fourth aspect of the present invention, there is provided a syringe prefilled 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 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 mg/ml, more typically in the range of from about 20-40mg/ml, most typically in the range of from about 22-38mg/ml.

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.

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 WO 98/44809 PCT/AU98/00226 14 turbid, non-immunogenic, injectable without blobbing 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 0 C, and'even when at a temperature of 10-12 0

C.

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 invitro produced, and is not limited to cross linked collagen but includes noncrosslinked collagen fibrils.

"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 is 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.

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 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 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 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 WO 98/44809 PCT/AU98/00226 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 HCI 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 of acetic acid with a small amount of concentrated fuming HCi 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 0 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 hours to weeks. At least 4 hours is preferred for digestion at lowered temperatures in the order of 5-15 0

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 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 and usually at around

C.

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 injectability through fine 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 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 WO 98/44809 PCT/AU98/00226 16 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 Sterile NaCI, typically in the form of a solution is added in accordance with the present invention with stirring, to the solution. The addition can occur at temperatures in the range of from about 4-25 0 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.

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.

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 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 0 C, usually around 20'C. The dialysis tubes preferably have a molecular weight cut off in the range of about 5,000-10,000. The concentration of the collagen in the dialysis tubes/bags 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 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, 32 or 33 gauge needle.

WO 98/44809 PCT/AU98/00226 17 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.

All non-self sterilising process fluids are sterile filtered The following equipment and products were used in the performance of the present invention: Sterile, pyrogen free water Clean Room Class C Laminar Air Flow cabinet Class A (LAF) Clean Room Attire Cool Room Scalpels and Blades Plastic Chopping Board BP Hypochlorite Solution Mincer (chemically sterilised with Hypochlorite) Carboys with sterile venting Waring Blender Disposable 0.2p capsule filters polypropylene filter cartridge in stainless steel housing BP glacial acetic acid BP Sodium Chloride USP Pepsin AR fuming hydrochloric acid Hydrated dialysis tubing (Gamma sterilised) Sterile funnel BP lignocaine HCI Disposable centrifuge bottles 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 hypochlorite solution.

4. The tendons are minced and the mince collected in the LAF.

WO 98/44809 PCT/AU98/00226 18 The mince is weighed and transferred to sterile carboys containing per each 100g of mince, 30 litres of 10% acetic acid, 20 ml of fuming HCI and Ig of Pepsin.

6. The mixture is allowed to digest for 24 hours.

7. The mixture is then blended in the Waring 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 50 filter and collected in a carboy.

9. 5M 'Sodium chloride solution is added with stirring to make the concentration 1M sodium chloride and the solution is chilled.

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.

The collagen gel is collected and the appropriate amount of lignocaine HCL solution is added to give the concentration of collagen as about 20-90mg/ml and the concentration of lignocaine HCI as about 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 Sample Bovine Collagen Batch Number Date Description (Odour) Odourless Description Translucent-clear gel mass Identification (SDA-PAGE) 2 major bands, due to collagen al and a 2[I] chains (monomers) are present. Higher molecular weight bands, due to collagen P3- (dimer) and y-chains (trimer) are also present.

WO 98/44809 PCT/AU98/00226 Identification (SDS-PAGE with 2 major bands, due to collagen al and a delayed reduction) 2[I] are present. No additional band due to al [III] chains (monomers) is present.

Higher molecular weight bands, due to collagen 13- (dimer) and y-chains (trimer) are also present.

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 present.

Transmittance at 430 nm) Forms clear solution as 0.5% solution in mM acetic acid.

pH (20 0 C, 0.5% in milliQ water) Arsenic <2 ppm Heavy Metals (as Pb) <5 ppm Amino acid analysis 11.5% hydroxyproline 0.45% tyrosine Concentration 2.6% by Kjeldahl nitrogen analysis 2.6% by amino acid analysis Pyrogen test (Rabbit) Passes.

Coliform Group Passes.

Total Bacterial Count Passes.

Claims (44)

1. 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.

2. An injectable collagen composition according to claim 1, said composition having a viscosity such that said composition is injectable through a 30 gauge needle without blobbing.

3. An injectable collagen composition according to claim 2 wherein said viscosity is such that said composition is injectable through a 30 gauge needle without blobbing when the composition is at a temperature in the range of from about 8 to 18 0 C.

4. An injectable collagen composition according to any one of claims 1-3 wherein said composition is substantially translucent to clear in appearance.

An injectable collagen composition according to any one of claims 1-4 wherein the pH of the composition is in the range of from about 3.5-5.5

6. An injectable collagen composition according to claim 5 wherein the pH of the composition is in the range of from about 4.2-4.8.

7. An injectable collagen composition according to any one of claims 1-4 wherein collagen concentration is in the range of from about 20-60 mg/ml.

8. An injectable collagen composition according to claim 7 wherein collagen concentration is in the range of from about 22-38mg/ml.

9. An injectable composition according to any one of claims 2-8 wherein said composition is injectable through a 30-35 gauge needle.

An injectable composition according to any one of claims 3-9 wherein the viscosity of the composition is such that it is injectable through a 30 gauge needle when the composition is at a temperature within the range of 10-15 0 C.

11. A process for the preparation of a collagen composition, said process comprising the steps of: mechanically processing mammalian connective tissue to form connective tissue fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 21

12. A process according to claim 11 wherein after said digesting step and before said mechanical shearing step the solution is mixed.

13. A process according to claim 11 wherein said mammalian connective tissue is bovine tendons.

14. A process according to claim 11 wherein said connective tissue fragments have both length and diameter in the range of from about 0.1-5mm.

A process according to claim 11 wherein said digestion occurs at a temperature range of from about 4-25 0 C.

16. A process according to claim 11 wherein the enzyme present in the acidic solution is selected from pepsin, trypsin and any other non-collagenase enzyme.

17. A process according to claim 11 wherein the digestion occurs for a time in the range of from about 4 to 28 hours.

18. A process according to claim 12 wherein said mixing is performed by blending said solution at a speed of from about 3 5 00-22,000rpm,

19. A process according to claim 12 wherein the mixed solution is chilled to a temperature of about 5-10 0 C for a period of up to about 24 hours before it is mechanically sheared.

A process according to claim 11 wherein the mechanical shearing of the mixed solution occurs through filtering the solution through at least one filter having a filter size range of from about 50-200 microns.

21. A process according to claim 20 wherein.the solution is filtered through a filter having a filter size of 50 microns.

22. A process according to claim 11 wherein the sheared solution is treated with an alkali metal non-phosphate salt solution sufficient to form a collagen suspension.

23. A process according to claim 22 wherein the alkali metal non-phosphate salt solution is sodium chloride solution. S

24. A process according to claim 11 wherein after said collagen suspension is formed, the solution is centrifuged in order to provide collagen gel. o.O

25. A process according to claim 24 wherein after said centrifuging to provide collagen gel, the collagen gel is collected, resuspended and the resuspended collagen is again centrifuged said to provide collagen gel. S..o

26. A process according to claim 11 wherein after said dialysis, an anaesthetic agent is added to the collagen to give an anaesthetic agent concentration of fromabout 2-

27. A process according to claim 26 wherein said anaesthetic agent is added to the collagen with mixing and is added to a final concentration of from about 3-5 mg/ml. 22

28. A process for the preparation of a collagen composition, said process comprising the steps of: mechanically processing bovine tendons under sterile conditions to form bovine tendon fragments of from about 0.1-5mm in both diameter and length, digesting said fragments in acidic solution containing pepsin at a temperature range of from about 4-25 0 C and over a period of about 24 hours, blending said solution at a speed of from about 3500-22,000rpm, 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, i) adding sodium chloride to said filtered solution to form a collagen suspension; ii) centrifuging the solution in order to provide collagen gel, collecting and resuspending the collagen gel and again centrifuging said resuspended collagen to provide collagen gel, dialysing the collagen gel against three changes of distilled water at 24 hour intervals to a collagen concentration of from about 20-90mg/ml, adding Lignocaine HCI solution to the collagen to give a Lidocaine concentration of from about 2-5mg/ml; and centrifuging to remove air from the collagen.

29. A process according to claim 11 wherein the process is performed under sterile conditions.

A process for the preparation of a collagen composition, said process comprising the steps of: mechanically processing mammalian connective tissue to form connective tissue fragments of from about 0.1-5mm in length, digesting said fragments in acidic solution containing at least one proteolytic (non collagenase) enzyme, mechanically shearing said solution such that substantially all collagen in the solution is capable of passing through a 200 micron filter, adding a non-phosphate salt and/or a non-phosphate buffer to said sheared solution to form a collagen suspension; and dialysing the collagen suspension to a collagen concentration of from about 20-90mg/ml substantially as hereinbefore described with reference to the Example.

31. A collagen composition produced according to a process as defined in any one of A claims 11-30. [R:\LIBA]3495.doc:eaa 23

32. A collagen composition according to claim 31 which is injectable, has a pH in the range of from about 3 to 6 and which is substantially translucent to clear in appearance.

33. 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 20-90mg/ml, substantially as hereinbefore described with reference to the Example.

34. A syringe prefilled with an injectable collagen composition according to any one of claims 1-10 or 31-33. o0

35. A method of augmenting soft tissue which comprises injecting a composition as claimed in any one of claims 1 to 10, or 31-33 to a patient in need thereof.

36. A method according to claim 35 wherein the augmentation includes the repair or correction of defects in soft tissue.

37. A method according to claim 36 wherein the defects include frown lines, nasolabial creases, wrinkles and other superficial sclerodermal lines.

38. A composition according to any one of claims 1 to 10, or 31 to 33 when used for augmenting soft tissue.

39. The composition when used according to claim 38 wherein the augmentation includes the repair or correction of defects in soft tissue.

40. The composition when used according to claim 39 wherein the defects include frown lines, nasolabial creases, wrinkles and other superficial sclerodermal lines.

41. Use of a collagen composition according to any one of claims 1 to 10 or 31 to 33 for the augmentation of soft tissue.

42. The use of claim 41 wherein the augmentation includes the repair or correction 25 of defects in soft tissue.

43. The use of claim 42 wherein the defects include frown lines, nasolabial creases, wrinkles and other superficial sclerodermal lines.

44. The use of claim 43 when used for augmenting soft tissue. DATED this Twenty first Day of November, 2000 ClearColl Pty Ltd Patent Attorneys for the Applicant SPRUSON FERGUSON *a [R:\LIBA 3495.doc:eaa