AU764712B2 - Apparatus for aseptic vortex flow concentration - Google Patents

Description

a

AUSTRALIA

Patents Act 1990 Matrix Pharmaceutical, Inc.

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Apparatus for aseptic vortex flow concentration The following statement is a full description of this invention including the best method of performing it known to us:- APPARATUS FOR ASEPTIC VORTEX FLOW

CONCENTRATION

Field of the Invention The invention relates generally to the aseptic processing of pharmaceutical and biological materials gels), more particularly to a bearing system for a vortex flow concentration/filtration apparatus. More specifically, the present 5 invention is directed to providing bearing surfaces in the concentration/filtration apparatus that do not shed or leave unwanted particles in the pharmaceutical or biological material being processed.

Background of the Invention In preparing pharmaceutical and biological materials for parenteral use, the material must be sterile and often must be concentrated. One method of concentrating these materials is by centrifugation. For viscous gels, centrifugation has associated problems of product recovery, aseptic operation and the maintenance of a closed system. Another method includes filtration. In conventional dead-end filtration with a stationary filter medium or membrane the liquid mixture flows perpendicular to the filter media. In tangential flow filtration, the liquid mixture passes tangentially past the filter media and the filtrate (permeate) passes through the medium. In such filtration, separation only occurs at the liquid-media boundary the boundary layer). The boundary layer tends to retain the filtered particles which are prevented from returning to the bulk solution. This leads to concentration polarization and in some cases to formation of gel layers on the filter media. Clogging or blinding of the filter media is a problem at any level of filtration, insofar as transmembrane flow flux) drops as the pores in the filter media become clogged. To eliminate 2 clogging and blinding, vortex flow filtration has been known to be used.

Vortex flow filtration devices typically employ a semi-permeable membrane as the filtration media. The vortex flow filtration apparatus relies on certain components of the filtered material being much more permeable through the membrane than other components. The purpose of the vortex flow filtration apparatus is to separate one or more substances by retaining some on one side of a membrane as a "retentate" while passing others through the membrane as a "permeate". In the processing of pharmaceutical and biological materials, often the concentrated retentate is the valuable portion and the permeate is drained off.

Vortex flow filtration uses a known hydrodynamic phenomenon to prevent clogging or blinding of the filter media caused by the accumulation of dissolved or suspended material thereon. The operation of these systems is discussed in U.S. Patent Nos. 4,790,942, 4,876,013, and 4,911,847 issued to ^Shmidt et al., which are incorporated herein by reference in their entirety.

In short, the vortex flow filtration device discussed in these references uses a membrane mounted on an inner body which rotates within a stationary outer body. The vortex flow apparatus prevents clogging by producing Taylor vortices in the parent fluid in the annular gap between the inner body and the stationary outer body. However, there is a need for a device that can effectively concentrate gels and/or semisolids, such as aqueous collagen dispersions, for parenteral use.

The known vortex flow filtration devices of the prior art are equipped with graphite bearings which are adequate for their intended purpose of separating cell culture or fermentation cells from the liquid content. These systems may be sterile but produce small levels of graphite particulates shed I 3 from the bearings, which is tolerable for separation purposes if the product produced is not intended for parenteral use.

Purified bovine collagen is used in a variety of medical devices including hemostats, sutures, corneal shields, and soft tissue augmentation. Collagen gels are often intermediates in the preparation of these devices and, in some cases, the gels represent the final medical products.

Sterile bovine collagen dispersions and gels with concentrations up to and higher are commercially available. These formulations are prepared by conventional processes whereby collagen is precipitated from solution and aseptically concentrated. A concentration/separation technique commonly employed is centrifugation. Centrifugation may require high capital expenditure; and presents sterilization and validation challenges and has product recovery problems for viscous, adhesive materials. Other separation techniques have also been found to be unsatisfactory. For example, conventional dead end 15 filtration and tangential flow filtration are not feasible because collagen fibers tend to clog or blind the filters.

Technical problems associated with concentrating collagen gels are attributable, in part, to their high viscosity and cohesive and adhesive properties.

For example, aqueous collagen products having about 0.3% (wt) to about 11% (wt) solids have viscosities ranging from about 30 mPa-sec to about 40,000 mPa-sec.

There is a need to provide an effective means for aseptically concentrating pharmaceutical and biological materials, including collagen and other gels and semisolids. The present invention offers such an apparatus for aseptic filtration/concentration of pharmaceutical and biological materials 4 (particularly semisolids and/or gels), such as aqueous dispersions of-collagen.

Summary of the Invention The present invention provides an apparatus for vortex flow concentration of semisolids or gels which has a shed-resistant bearing interface so that undesirable particles do not shed off the bearing and discolor and/or contaminate the pharmaceutical or biological material being processed for parenteral use. The apparatus of the present invention is in response to the discovery that known vortex flow filtration systems having graphite bearings cannot be used to concentrate parenteral products because of the graphite particles that shed from :e o 10 the bearings. The present invention allows for the first time the use of vortex flow filtration to concentrate disperse compositions dilute suspensions) into semisolids or gels for parenteral use.

In one embodiment of the present invention, there is provided an apparatus having a permeable or semi-permeable. membrane for aseptic vortex flow concentration of a dispersion to result in a gel or semisolid. The apparatus comprises a rotatable body having an outer side wall, an upper spindle,. and a lower spindle, an outer body having an upper end with openings therein, a lower end with openings therein, and an inner side wall spaced from the outer side wall of the rotatable body to define an annular space for receiving the permeable or semi-permeable membrane. There is also a shed-resistant upper spindle support in the upper end of the outer body for receiving the upper spindle and a shedresistant lower spindle support in the lower end of the outer body for receiving the lower spindle. There is provided means for rotating the rotatable body about an axis through the upper spindle and the lower spindle at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotatable body and the permeable or semi-permeable membrane. The shedresistant upper spindle support is located adjacent to the openings in the upper end and the shed-resistant lower spindle support is located adjacent to the openings in the lower end such that the dispersion passes through the openings and lubricates the shed-resistant upper spindle support and the shed-resistant lower spindle support.

In another embodiment of the present invention, there is provided an apparatus having a filter for aseptic vortex flow concentration of a dispersion to result in a gel or semi-solid. The apparatus comprises a housing having an upper end with an outlet therein, a lower end with an inlet therein and an inner side wall. An upper rotor support is located in the upper end of the housing and a lower rotor support is located in the lower end of the housing. There is a rotor with an upper shed-resistant member and a lower shed-resistant member rotatably received in the upper rotor support and the lower rotor support. The rotor has an outer side wall angularly spaced from the inner side wall of the housing to define an annular space for receiving the filter. There is provided means for rotating the rotor about an axis through the center of the rotor at a rate sufficient to produce Taylor vortices in an annular space between the outer side wall of the rotor and the filter. The upper rotor support is located adjacent to the outlet in the upper end and the lower rotor support is located adjacent to the inlet in the lower end such that the dispersion passes over the upper shed-resistant member and the lower shed-resistant member.

In an aspect, the present invention provides an apparatus having a permeable or semi-permeable membrane for aseptic vortex flow concentration of a dispersion, including: a rotatable body having an outer side wall, an upper spindle, and a lower spindle; an outer body having an upper end with openings therein, a lower end with openings therein, and an inner side wall spaced from the outer side wall of the rotatable body to define an annular space for receiving the permeable or semi-permeable membrane; an upper spindle support in the upper end of the outer body for receiving the 30 upper spindle for producing a shed-resistant interface; a lower spindle support in the lower end of the outer body for receiving the lower spindle for producing a shed-resistant interface; and goes means for rotating the rotatable body about an axis through the upper spindle and the lower spindle at a rate sufficient to produce Taylor vortices in an annular gap 35 between the outer side wall of the rotatable body and the permeable or semi-permeable membrane; said upper spindle support being located adjacent to the openings in the upper end and the lower spindle support being located adjacent to the openings in the lower end such that the dispersion passes through the openings and lubricates the upper spindle support and the lower spindle support.

In another aspect, the present invention provides an apparatus having a filter for aseptic vortex flow concentration of a dispersion including: a housing having an upper end with an outlet therein, a lower end with an inlet therein, an inner side wall, an upper rotor support in the upper end of the housing, and a lower rotor support in the lower end of the housing; a rotor having an upper shed-resistant member and a lower shed-resistant member rotatably received in the upper rotor support and the lower rotor support, the rotor having an outer side wall spaced from the inner side wall of the housing to define an annular space for receiving the filter; and means for rotating the rotor about an axis through the center of the rotor at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotor and the filter; said upper rotor support being located adjacent to the outlet in the upper end and the lower rotor support being located adjacent to the inlet in the lower end such that the dispersion passes over the upper shed-resistant member and the lower shed-resistant member; an exit port located in said housing; and .said inlet in the lower end and the outlet in the upper end of the housing are in fluid communication with the annular gap between the outer side wall of the rotor and 25 the filter such that the dispersion enters the annular gap and the dispersion is concentrated in the annular gap as a permeate passes through the filter and exits through the exit port.

In yet a further embodiment of the present invention, there is provided a process for separating a collagen composition into a permeate portion and a concentrated 30 retentate portion, which includes the steps of: providing a vortex flow filtration apparatus having an inlet and an outlet: a housing having an upper end with an outlet therein, a lower end with an inlet :*oooe o therein, an inner side wall, an upper rotor support in the upper end of the housing, and a lower rotor support in the lower end of the housing; 0* a rotor having an upper shed-resistant member and a lower shed-resistant member rotatably received in the upper rotor support and the lower rotor support, the rotor having an outer side wall spaced from the inner side wall of the housing to define an annular space for receiving the filter; and means for rotating the rotor about an axis through the center of the rotor at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotor and the filter; said upper rotor support being located adjacent to the outlet in the upper end and the lower rotor support being located adjacent to the inlet in the lower end such that the dispersion passes over the upper shed-resistant member and the lower shed-resistant member; an exit port located in said housing; and said inlet in the lower end and the outlet in the upper end of the housing are in fluid communication with the annular gap between the outer side wall of the rotor and the filter such that the dispersion enters the annular gap and the dispersion is concentrated in the annular gap as a permeate passes through the filter and exits through the exit port; causing the collagen composition to flow through the inlet and into the gap; and rotating the rotor means at a speed effective to separate the collagen composition into permeate and concentrated retentate portions.

In yet a further aspect, the present invention provides a process for producing a S concentrated collagen composition from a collagen suspension, which includes the steps S. 25 of: S providing a vortex flow filtration apparatus having an inlet and an outlet including: a housing having an upper end with an outlet therein, a lower end with an inlet therein, an inner side wall, an upper rotor support in the upper end of the housing, and a lower rotor support in the lower end of the housing; a rotor having an upper shed-resistant member and a lower shed-resistant member rotatably received in the upper rotor support and the lower rotor support, the rotor having an outer side wall spaced from the inner side wall of the housing to define an annular space for receiving the filter; and 6b means for rotating the rotor about an axis through the center of the rotor at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotor and the filter; said upper rotor support being located adjacent to the outlet in the upper end and the lower rotor support being located adjacent to the inlet in the lower end such that the dispersion passes over the upper shed-resistant member and the lower shed-resistant member; an exit port located in said housing; and said inlet in the lower end and the outlet in the upper end of the housing are in fluid communication with the annular gap between the outer side wall of the rotor and the filter such that the dispersion enters the annular gap and the dispersion is concentrated in the annular gap as a permeate passes through the filter and exits through the exit port; causing the collagen suspension to flow through the inlet and into the gap; rotating the rotor means at speeds effective to separate the collagen suspension into a permeate and a retentate; recirculating at least a portion of the retentate into the fluid gap for further separation into permeate and retentate; and continuing steps and until the retentate has a collagen concentration of between about 3% to about 15% The process of the present invention is based, in part, on the employment of vortex flow filtration to separate collagen suspensions to form highly concentrated, :viscous compositions. The inventive process is capable of aseptically concentrating collagen dispersions without the fouling and other problems associated with prior art 25 methods. Specifically, the inventive process can produce sterile collagen compositions having about 0.25% to about 12% collagen; it is expected the process can produce compositions having up to 15% collagen. (Except as specifically noted, all percentages referred to herein shall be on a weight basis.) The process can aseptically concentrate any type of collagen from any animal although the process will be described employing experimental data from bovine collagen.

Brief Description of the Drawings V Many objects and advantages of the present invention will be apparent to those of ordinary skill in the art when this specification is read in conjunction with the attached 35 drawings wherein like reference numerals are applied to like elements and wherein: 35 drawings wherein like reference numerals are applied to like elements and wherein: FIG. 1 is a diagrammatical view of a system for aseptically filtering or concentrating a pharmaceutical or biological material in accordance with one embodiment of the present invention; 00 e FIG. 2 is a sectional view of a vortex flow filtration/concentration apparatus in accordance with one embodiment of the present invention; FIG. 3 is a top plan view of a spindle support in accordance with one embodiment of the present invention for use in the apparatus shown in FIG.

2; FIG. 4 is a top plan view of a spindle in accordance with one embodiment of the present invention for use in the apparatus shown in FIG.

2; FIG. 5 is a top plan view of a bearing support in accordance with one embodiment of the present invention for use in the apparatus shown in FIG.

15 2; and FIG. 6 is a sectional view of a vortex flow filtration/concentration apparatus in accordance with one embodiment of the present invention.

Detailed Description of the Preferred Embodiments The present invention will be described in detail below, but first a few terms will be defined.

Definitions COLLAGEN with respect to sterile collagen compositions prepared by the present invention shall include, but is not limited to, naturally occurring collagen and, more preferably, atelopeptide collagen derived by the removal of the nonhelical terminal portions of the native collagen molecule.

COLLAGEN COMPOSITION generally refers to aqueous collagen formulations comprising up to about 15% collagen. Collagen compositions range from dilute solutions wherein the collagen molecules are solubilized, to thick, viscous semisolids The present invention can be employed to produce collagen compositions having up to about 15% collagen. It is understood that sterile collagen compositions produced by the present invention may contain buffering salts and other non-collagen solutes or suspended materials.

The viscosity of collagen composition will depend on a number of factors including collagen concentration, pH, temperature, salt content and the relative proportion of collagen monomers to oligomers present. The inventive process is capable of producing concentrations of 12% or more by I* weight which have corresponding viscosities of about 35,000 mPa.sec and higher. At ambient temperatures, collagen generally is soluble at about pH 2 as single molecules comprised of a triple helix and having a molecular weight of about 300 kDa. Small oligomers dimers, trimers, etc.) are also soluble under these conditions. At pH of about 4.5 to 4.8 or above, the collagen molecules assemble into fibrils and/or large fibers that precipitate out of solution. Dispersions of fibrillar collagen generally exhibit pseudoplastic (or shear-thinning) behavior so that their viscosities decrease with increasing rate of shear. Preferably, the collagen compositions produced comprise about 3% to about 12% collagen, more preferably about 4% to about and most preferably about 6% to about 8%.

COLLAGEN SOLUTION generally refers to collagen compositions comprising about 0.1% to about 0.3% (wt) collagen wherein the collagen is solubilized; such compositions typically have viscosities ranging from about mPa.sec to about 50 mPa.sec at ambient temperature. Collagen solutions are generally optically clear and freely flowable.

COLLAGEN SUSPENSION generally refers to collagen composition comprising about 0.1% to about 1% collagen wherein the collagen is only partially solubilized, i.e. a significant portion of the protein exists as solidlike particles fibrils) dispersed in the liquid medium. These compositions are generally not optically clear.

COLLAGEN GEL OR GEL generally refers to collagen compositions comprising about 1% to about 8% collagen and have viscosities ranging from about 1,000 mPa.sec to about 30,000 mPa.sec or higher. The consistency of the collagen gel can range from being partially free flowing to a viscous semisolid or paste. The gel comprises a mixture of solubilized collagen i molecules and predominantly insoluble collagen solids fibrils). It is understood that the terms "collagen solution", "collagen suspension" and "collagen gel" do not necessarily represent distinct forms of collagen compositions, but rather, they are convenient means for describing collagen compositions at different concentrations, pHs and/or temperatures.

CLOSED SYSTEM generally describes an apparatus that serves as a 20 barrier between the product and the ambient environment. Once sterilized, a ooooa closed system is capable of aseptically processing materials such that the starting materials as well as the intermediate and final products remain sterile. Aseptic processing is used when the final product cannot be sterilized with heat, chemical agents, or other means. Aqueous collagen compositions are examples of such products. Closed systems provide additional assurance that the process can be conducted aseptically, and are therefore particularly well suited for aseptic processing of material like collagen solutions, suspensions, and gels.

GEL refers to colloids in which a dispersed solid or polymeric phase has combined with a continuous phase to produce a viscous product.

SHED-RESISTANT refers to materials which do not produce particulates to the extent that the product in contact with those materials is contaminated with those particulates. Acceptable levels of particulate matter in injections is discussed in USP XXIII <788>. Examples of shed-resistant materials include but are not limited to materials which will operate in continuous non-lubricated service with less than 500 microinches of wear at 10,000 PV for 40 hours where PV speed (ft/min) x load (psi) and the mating surface is 316 stainless steel; continuous non-lubricated 10 service with less than 400 microinches of wear at 10,000 PV for 40 hours 4* *4 where the mating surface is 1018 stainless steel; and/or continuous nonlubricated service with less than 350 microinches of wear at 10,000 PV for hours where the mating surface is 303 stainless steel.

In addition, or as an alternative, to the requirements just discussed, the following procedure can be conducted to determine if the material is sufficiently shed-resistant.

Clean, pure, particulate free water or product is recycled through the thoroughly cleaned system for the maximum proposed run time. The water is then tested for particulates according to USP XXIII <788>.

Also see the procedures described in USP XXIII, <788> "Particulate matter in Injections" which is incorporated herein by reference in its entirety.

CLASS VI MATERIAL refers to materials classified as USP Class VI as defined in USP XXIII, <88> "Biological Reactivity Tests, In Vivo" which is incorporated herein by reference in its entirety.

Three tests are applied in U.S.P. XXIII, <88> to the materials: the Systemic Injection Test, Intracutaneous Test, and Implantation Test. These three tests are reproduced below directly from U.S.P. XXIII, For the purposes of these tests, these definitions apply. The "sample" is the specimen under test or an extract prepared from such a specimen. A "blank" consists of the same quantity of the same extracting medium that is used for the extraction of the specimen under test, treated in the same manner as the extracting medium containing the specimen under test. A "negative control" is a specimen that gives no reaction under the conditions of the test. Materials are classified on the basis of the response criteria prescribed in the following Table I.

Table 1. Classification of Plastics.

S

*5

S

S.

S.

S

S

Plastic Classesa Conducted Tests To Be 10 II I1IV V VI Test Material X x x X X X Extract of Sample xxx in Sodium x x x Chloride Injection Animal Dose Mouse 50 mL/kg Rabbit 0.2 mL/animal at each of sites Procedure A (iv)

B

x x x x x Extract of Sample Mouse 50 mL/kg A (iv) x x x x in1 in 20 Solution Rabbit 0.2 mL/animal B of Alcohol in at each of Sodium Chloride 10 sites Injection x x x Extract of Sample Mouse 10 g/Kg A (ip) x x in Polyethylene Rabbit 0.2 mL/animal B Glycol 400 at each of sites x x x x Extract of Sample Mouse 50 g/Kg A (ip) x x x in Vegetable Oil Rabbit 0.2 mL/animal B at each of sites x x Implant strips of Rabbit 4 strips/animal C Sample Tests required for each class are indicated by in appropriate columns.

b Legend: A (ip) Systemic Injection Test (intraperitoneal); A (iv) Systemic Injection Test (intravenous); B Intracutaneous test (intracutaneous); C Implantation Test (intramuscular implantation).

1, 12 Apparatus The apparatus for the tests includes the following: AUTOCLAVE Use an autoclave capable of maintaining a temperature of 121 2.0* equipped with a thermometer, a pressure gauge, a vent cock, a rack adequate to accommodate the test containers above the water level, and a water cooling system that will allow for cooling of the test containers to about, but not below, 20* immediately following the heating cycle.

*0 0 S: OVEN Use an oven, preferably a forced-circulation model, that will maintain operating temperatures of 500 or 70° within p o EXTRACTION CONTAINERS Use only containers, such as ampuls or 10 screw-cap culture test tubes, of Type I glass. If used, culture tests tubes are f closed with screw caps having suitable elastomeric liners. The exposed surface of the elastomeric liner is completely protected with an inert solid disk 0.05 mm to 0.075 mm in thickness. A suitable disk may be fabricated from a polytef resin.

Preparation of Apparatus Cleanse all glassware thoroughly with chromic acid cleansing mixture, or if necessary with hot nitric acid, followed by prolonged rinsing with water. Clean cutting utensils by an appropriate method successive cleaning with acetone and methylene chloride) prior to use in subdividing a specimen. Clean all other equipment by thorough scrubbing with a suitable detergent and prolonged rinsing with water.

Render containers and equipment used for extraction, and in transfer and administration of test material, sterile and dry by a suitable process. [Note-If ethylene oxide is used as the sterilizing agent, allow adequate time for complete degassing.] Extracting Media- SODIUM CHLORIDE INJECTION. Use Sodium chloride Injection containing 0.9% of Sodium Chloride NaCL.

1 in 20 Solution of Alcohol in Sodium Chloride Injection.

Polyethylene Glycol 400.

Vegetable Oil Use freshly refined Sesame Oil or Cottonseed Oil or other suitable vegetable oils.

0 Drug Product Vehicle (where applicable).

10 Water For Injection.

[Note The Sesame Oil or Cottonseed Oil or other suitable vegetable oil meets the following additional requirements. Obtain, if possible, freshly refined oil. Use three properly prepared animals and inject the oil intracutaneously in a dose of 0.2 mL into each of 10 sites per animal, and observe the animals at 24, 15 48, and 72 hours following injection. Rate the observations at each site on the numerical scale indicated in Table 5. For the 3 rabbits (30 injection sites), at any 0 observation time, the average response for erythema is not greater than.0.5 and for edema is not greater than 1.0 and no site shows a tissue reaction larger than mm in overall diameter. The residue of oil at the injection site should not be misinterpreted as edema. Edematous tissue blanches when gentle pressure is applied.] Procedure Preparation of Sample- Both the Systemic Injection Test and the Intracutaneous Test may be performed using the same extract, if desired, or separate extracts may be made for each test. Select and subdivide into portions a Sample of the size indicated in Table 2. Remove particulate matter, such as lint and free particles by treating each subdivided Sample or Negative Control as follows: place the Sample into a clean, glass-stoppered 100-mL graduated cylinder of Type I glass, and add about 70 mL of Water for Injection. Agitate for about 30 seconds, and drain off the water, repeat this step, and dry those pieces prepared for the extraction with Vegetable Oil in an oven at a temperature not exceeding 50*. [Note Do not clean the Sample with a dry or wet cloth or by rinsing or washing with an organic solvent surfactant, etc.] Table 2. Surface Area of Specimen To Be Used' Amount of Sample for Each 20 mL of Form of Thickness Extracting Medium Subdivided Into Material Film or <0.5 mm Equivalent of 120 cm 2 Strips of about 5 X sheet total surface area (both 0.3 cm sides combined) to 1 mm Equivalent of 60 cnm total surface area (both sides combined Tubing <0.5mm Length (in cm) 120 Sections of about (wall) cm 2 /(sum of ID and OD X 0.3 cm circumferences) Length (in cm) to 1 mm cm 2 /(sum of ID and OD S(wall) circumferences) Slabs, 1 mm Equivalent of 60 cm 2 Pieces up to about tubing, and total surface area (all X 0.3 cm molded exposed surfaces items combined) Elastomers 1 mm Equivalent of 25 cm 2 Do not subdivide 2 total surface area (all exposed surfaces combined) 1When surface area cannot be determined due to the configuration of the specimen, use 0.1 g of elastomer or 0.2 g of plastic or polymers for every 1 mL of extracting fluid.

2 Molded elastomeric closures are tested intact.

Preparation of Extracts Place properly prepared Sample to be tested in an extraction container, and add 20 mL of the appropriate extracting medium.

Repeat these directions for each extracting medium required for testing. Also prepare one 20-mL blank of each medium for parallel injections and comparisons. Extract by heating in an autoclave at 121" for 60 minutes, in an oven at 70' for 24 hours, or at 50* for 72 hours. Allow adequate time for the liquid within the container to reach the extraction temperature.

Note The extraction conditions should not in any instance cause physical changes such as fusion or melting of the Sample pieces, which result in a decrease in the available surface area. A slight adherence of the pieces can be tolerated. Always add the cleaned pieces individually to the extracting medium.

If culture tubes are used for autoclave extractions with Vegetable Oil, seal screw caps adequately with pressure-sensitive tape.

Cool to about room temperature but not below 20°, shake vigorously for several minutes and decant each extract immediately, using aseptic precautions into a dry, sterile vessel. Store the extracts at a temperature between 20° and and do not use for tests after 24 hours. Of importance are the contact of the extracting medium with the available surface area of the plastic and the time and temperature during extraction, the proper cooling, agitation, and decanting process, and the aseptic handling and storage of the extracts following extraction.

Systemic Injection Test This test is designed to evaluate systemic responses to the extracts of materials under test following injection into mice.

Test Animal Use healthy, not previously used albino mice weighing 16 between 17 and 23 gramnS. For each test group use only mice Of the same source.

Allow water and food, commonly used for laboratory animals and of known composition,~ ad libitumn.

Procedure [Note: Agitate each extract vigorously prior to withdrawal of injection doses to ensure even distribution of the extracted matter. However, visible particulates should not be injected intravenously.] Inject each of the five mice in a test group with the Sample or the Blank as outlined in Table 3, except to dilute each gram of the extract of the Sample prepared with polyethylene .0.0 Glycol 400, and the corresponding blank, with 4.1 volumes of Sodium Chlornde Injection to obtain a solution having a concentration of about 200 mg of polyethylene glycol per niL.

Observe the animals immediately after injection, again 4 hours after injection, and then at least at 24, 48, and 72 hours. If during the observation period none of the animals treated with the extract .of the Sample shows a significantly greater biological reactivity than the animals treated with the Blank, the Sample meets the requirements of this test. If two or more mice die, or if abnormal behavior such as convulsions or prostration occurs in two or more mice, or if a body weight loss greater than 2 grams occurs in three or more mice, the Sample does not meet the requirements of the test. if any animals treated with the Sample show only slight signs of biological reactivity, and not more than one animal shows gross symptomis of biological reactivity or dies, repeat the test using groups of 10 mice. On the repeat test, all 10 animals treated with the Sample show no significant biological reactivity above the Blank animals during the observation period.

17 Table 3. Injection Procedure-Systemic Injection Test.

Injection Rate, /L per Extract or Blank Dose per kg Route" second Sodium Chloride Injection 50 mL IV 100 1 in 20 solution of 50 mL IV 100 Alcohol in Sodium Chloride Injection Polyethylene Glycol 400 10 g IP Drug product vehicle 50 ml IV 100 (where applicable) 50 mL IP Vegetable Oil 50 mL IP *5 'IV intravenous (aqueous sample and blank); (oleaginous sample and blank).

IP intra-peritoneal Intracutaneous Test This test is designed to evaluate local responses to the extracts of material under test following intracutaneous injection into rabbits.

Test Animal Select healthy, thin-skinned albino rabbits whose fur can be clipped closely and whose skin is free from mechanical irritation or trauma. In handling the animals, avoid touching the injection sites during observation periods, except to discriminate between edema and an oil residue. [Note Rabbits previously used in unrelated tests and that have received the prescribed rest period, may be used for this test provided that they have clean, unblemished skin.] Procedure [Note Agitate each extract vigorously prior to withdrawal of injection doses to ensure even distribution of the extracted matter.] On the day of the test, closely clip the fur on the animal's back on both sides of the spinal column over a sufficiently large test area. Avoid mechanical irritation and )A 18 trauma. Remove loose hair by means of vacuum. If necessary, swab the skin lightly with diluted alcohol, and dry the skin prior to injection. More than one extract from a given material can be used per rabbit, if you have determined that the test result will not be affected. For each Sample use two animals and inject each intracutaneously, using one side of the animal for the Sample and the other side for the Blank as outlined in Table 4. [Note Dilute each gram of the extract of the Sample prepared with Polyethylene Glycol 400, and the corresponding Blank, with 7.4 volumes of Sodium Chloride Injection to obtain a solution having a concentration of about 120 mg of polyethylene glycol per mL.] 9* 9 Examine injection sites for evidence of any tissue reaction such as erythema, edema, and necrosis. Swab the skin lightly, if necessary, with diluted eee.* alcohol to facilitate reading of injection sites. Observe all animals at 24, 48, and 72 hours after injection. Rate the observations on a numerical scale for the 15 extract of the Sample and for the Blank using Table 5. Reclip the fur as necessary during the observation period. The average erythema and edema scores for Sample and Blank sites are determined at every scoring interval (24, 48, and 72 hours) for each rabbit. After the 72 hour scoring, all erythema scores plus edema scores are totalled separately for each Sample and Blank. Divide each of the totals by 12 (2 animal X 3 scoring periods X 2 scoring categories) to determine the overall mean score for each Sample versus each corresponding Blank. The requirements of the test are met if the difference between the Sample and the Blank mean score is 1.0 or less. If at any observation period the average reaction to the Sample is questionably greater than the average reaction to the S Blank, repeat the test using three additional rabbits. The requirements of the test are met if the difference between the Sample and the Blank mean score is 1.0 or less.

19 Table 4. Intracutaneous Test.

Extract or Number of Sites Dose Blank (per animal) L per site Sample 5 200 Blank 5 200 Table 5. Evaluation of Skin Reactions.

Erythema and Eschar Formation Score No erythema 0 Very slight erythema (barely perceptible) 1 Well-defined erythema 2 Moderate to severe erythema 3 *:Severe erythema (beet-redness) to slight eschar formation (injuries in depth) 4 Edema Formation' Score No edema 0 Very slight edema (barely perceptible) 1 Slight edema (edges of area well defined by definite raising) 2 S• Moderate edema (raised approximately 1 3 mm) Severe edema (raised more than 1 mm 4 and extending beyond the area of exposure) Excludes noninflammatory (mechanical) edema from the blank or extraction fluid.

Implantation Test The implantation test is designed for the evaluation of plastic materials and other polymeric materials in direct contact with living tissue. Of importance are the proper preparation of the implant strips and their proper implantation under aseptic conditions. Prepare for implantation 8 strips of the Sample and 4 strips of U.S.P. Negative Control Plastic RS. Each strip should measure not less than 10 X 1 mm. The edges of the strips should be as smooth as possible to avoid additional mechanical trauma upon implantation. Strips of the specified minimum size are implanted by means of a hypodermic needle (15-to-19 gauge) with intravenous point and a sterile trocar. Use either presterilized needles into which the sterile plastic strips are aseptically inserted, or insert each clean strip into a needle, the cannula and hub of which are protected with an appropriate cover, and then subjected to the appropriate sterilization procedure. [Note Allow for proper degassing if agents such as ethylene oxide are used.] Test Animal Select healthy, adult rabbits weighing not less than 2.5 kg, 10 and whose paravertebral muscles are sufficiently large in size to allow for implantation of the test strips. Do not use any muscular tissue other than the paravertebral site. The animals must be anesthetized with a commonly used anesthetic agent to a degree deep enough to prevent muscular movements, such as twitching.

Procedure Perform the test in a clean area. On the day of the test or up to 20 hours before testing, clip the fur of the animals on both sides of the spinal column. Remove loose hair by means of vacuum. Swab the skin lightly with diluted alcohol and dry the skin prior to injection.

Implant four strips of the Sample into the paravertebral muscle on one side of the spine of each of 2 rabbits, 2.5 to 5 cm from the midline and parallel to the spinal column, and about 2.5 cm apart from.each other. In a similar fashion implant 2 strips of U.S.P. Negative Control Plastic RS in the opposite muscle of each animal. Insert a sterile stylet into the needle to hold the implant strip in the tissue while withdrawing the needle. If excessive bleeding is observed after implantation of a strip, place a duplicate strip at another site.

Keep the animals for a period of not less than 120 hours, and sacrifice them at the end of the observation period by administering an overdose of an anesthetic agent or other suitable agents. Allow sufficient time to elapse for the tissue to be cut without bleeding. Examine macroscopically the area of the tissue surrounding the center portion of each implant strip. Use a magnifying lens and auxiliary light source. Observe the Sample and Control implant sites for hemorrhage, necrosis, discolorations, and infections, and record the observations. Measure encapsulation, if present, by recording the width of the capsule (from the periphery of the space occupied by the implant Control or 10 Sample to the periphery of the capsule) rounded to the nearest 0.1 mm. Score encapsulation according to Table 6.

Calculate the differences between average scores for the Sample and Control sites. The requirements of the test are met if the difference does not exceed 1.0 or if the difference between the Sample and Control mean scores for more than one of the four implant sites does not exceed 1 for any implanted animal.

Table 6. Evaluation of Encapsulation in the Implantation Test Capsule Width Score None 0 up to 0.5 mm 1 0.6 1.0 mm 2 1.1 2.0 mm 3 Greater than 2.0 mm 4 Apparatus 10 of the present invention is particularly useful for the aseptic concentration of pharmaceutical and biological gels and excipients for parenteral use, in particular aqueous collagen gels and other protein dispersions. For collagen to be incorporated into parenteral pharmaceutical or biological formulations it must be sterile. Collagen gels are often intermediates in the preparation of parenteral pharmaceutical or biological formulations, or sometimes the final medical products themselves. Often after a bioburden reduction step, there is a need to precipitate and aseptically concentrate the collagen. Apparatus can be used to concentrate collagen from 0.25 by weight suspensions to greater than 12% by weight gels. The apparatus concentrates collagen at a high flux rate through a membrane and with a low transmembrane pressure by utilizing a three-dimensional flow profile, known as Taylor flow or Taylor 10 vortices, to keep the membrane surface from fouling, blinding, clogging, etc.

Taylor, using a framework of linear theory and considering viscous fluids, found that when a certain Taylor's number was exceeded, axially circumferential vortices appear which rotate in alternately opposing directions.

S. Taylor determined that the minimum condition for the establishment of such vortices, defined as the Taylor number is xd d T V, 41.3 where v is the kinematic viscosity of the fluid, is the peripheral velocity of the inner rotating cylinder, R, is the radius of an inner rotating cylinder, and d is the dimension of the annular gap filled with fluid between the inner cylinder and a stationary outer cylinder.

Taylor and others determined that the vortices would persist in some cases at T, 400 and in other cases up to T, 1700, but that turbulence would ensue if the Reynolds number rose above about 1000. The Reynolds number is o(2d) R= v where o is the axial velocity. Those skilled in hydrodynamics will appreciate that a time average velocity profile of fluid flow will generate a smooth curve, 5 but an instantaneous velocity profile is very jagged. Thus, the Taylor vortices may be characterized.as the main flow, but there will be a turbulent component and, as T, rises, this instantaneous turbulent velocity will ultimately become more important.

In one embodiment of the vortex flow concentrating device of the present 10 invention, the outer stationary cylinder is a permeable or semi-permeable membrane. The axially circumferential vortices that rotate in alternately opposing directions are formed between the rotating cylinder and the inside wall o of the membrane. The strength of the vortices is directly proportional to the rotation rate of the rotating cylinder causing the permeate flow to increase with rotor speed. Because there is a net axial velocity due to the feeding and removal of fluid into the device, the individual vortices assume what appears to be a helical shape and move from the inlet to the outlet of the device. The rotation of the individual vortices and the movement up the inside wall of the membrane continuously scours the inside of the membrane so that gels, particulates and colloids that would otherwise collect there are pulled back into the fluid. When the conditions for establishing the Taylor vortices are met, the filtration/concentration apparatus operates at very minimal transmembrane pressures (approximately less than 3 psi).

Apparatus 10 can be used in a sterile, closed loop system 14 such as shown in FIG. 1. System 14 is shown as one possible embodiment, other elements can be present such as a heat exchanger on the return to the reservoir to remove heat generated by the rotating cylinder and the pump, or system 14 can be a subsystem of a larger system. A pharmaceutical or biological formulation, such as an aqueous dispersion of collagen, is placed in reservoir 12. The aqueous collagen dispersion begins as a free flowing slurry with a concentration of approximately 0.25% and a viscosity of approximately 10 to 15 mPa.sec. The collagen dispersion is circulated through system 14 with pump 16. For example, pump 16 can be a low shear 10 peristaltic pump or lobe pump. Pump 16 pushes the aqueous collagen into inlet 18. The aqueous collagen dispersion passes through apparatus 10 (as will be described in more detail later) and exits through outlet 20. Water and soluble components (permeate) pass through the semi-permeable membrane and are removed from one or both drains 60, 61. The collagen dispersion is 15 then recirculated through system 14 with pump 16 until the desired S.concentration is achieved. A related co-pending application which further describes the closed loop system is Serial No. 08/742,677 filed on October 31, 1996 (now US Patent No. 5,874,006) and entitled "Aseptic Collagen Concentration Process" which application is incorporated herein in its entirety.

The device illustrated in FIGS. 1 through 5 employs a rotating body housed within an outer stationary cylinder that is a permeable or semipermeable membrane. Vortex flow filtration devices suitable for the present invention may also comprise devices, for instance, wherein the membrane is mounted on an inner body which rotates within a stationary outer body. See, for instance, U.S. Pat. Nos. 4,790,942, 4,876,013, and 4,911,847. In these devices, Taylor vortices developed in the parent fluid in the annular gap between the inner body and the stationary outer body reduces blinding. As shown in FIG. 6, which is a partial sectional view of such a device, an inner, rotatable body 100 comprising a permeable or semi-permeable membrane 102, is positioned within an outer stationary 101 body having an inner side wall 103. The inner body has an outer side wall spaced from the inner side wall of the stationary body which define an annular gap 105 which is in communication with the devices inlet.

In operation, as the inner rotatable body is rotated, material is pumped into the annual gap. The inner rotatable body with the membrane is rotated at a sufficient rate to produce Taylor vortices in the annular space so as to 10 cause permeate to flow into the inner region of the inner body, while the retentate remains in the annular gap.

As is apparent, for vortex flow filtration device employed in the inventive process, the speed of rotation of the rotatable body and the width of the annular gap are two parameters that can be optimized. Other parameters include permeate rates, retentate recirculation rates, and transmembrane pressures.

The apparatus of the present invention allows for the concentration of collagen suspensions over a continuum from about 0.25% to greater than 12% by weight. Collagen dispersions at or above 2% by weight concentration are essentially a semisolid or a gel. Typically, a 2% collagen dispersion will have a viscosity of approximately 1,000 mPa.sec or above. Through the novel features discussed below, the present invention is capable of producing concentrations of 12% or more by weight and processing semisolids or gels having a viscosity of approximately 35,000 mPa.sec or more. Therefore, the present invention provides an apparatus that is capable of concentrating gels and semisolids of a large variety of pharmaceutical and biological materials under sterile conditions.

Apparatus 10 can be used to concentrate a thin slurry to produce a semisolid or to concentrate a gel or semisolid to produce a higher concentration semisolid.

To better understand the novel features of thle present invention, apparatus will now be described in greater detail with respect to FIG. 2. Pump 16 pushes the aqueous pharmaceutical or biological material, such as a collagen dispersion, into inlet 18. The pharmaceutical or biological material enters inlet 18 and flows through openings .22 and center opening 23 in lower support 24.

Lower support 24 and inlet 18 are attached to apparatus 10 by fasteners 26. As will be recognized by one of ordinary skill in the art other fasteners can be used.

Fasteners 26 are advantageous, however, because they are removable which allows for ease in cleaning and sterilization as well as repair of the inlet, apparatus, and support. Lower support 24 holds lower spindle support 28.

Lower spindle support 28 has an opening 32 for receiving lower spindle 34 of rotatable body or rotor 30. Opening 32 also allows the material flowing through center opening 23 to flow over the lower spindle 34 to help lubricate the surfaces S• on the lower spindle support 28 and lower spindle 34. Similarly, at the other end of apparatus 10, support 25 holds upper spindle support 29. Upper spindle support 29 has an opening 33 for receiving upper spindle 36 of rotatable body Upper spindle support 29 is the same as lower spindle support 28. Upper spindle 36 and lower spindle 34 can be threaded and epoxied into rotatable body 30, be integrally formed with the rotatable body or attached by other known means.

In one embodiment, lower spindle support 28 in FIG. 2 and 3 is a bushing. Lower spindle pin 38 and upper spindle pin 40 are highly polished hard chrome plated 316 stainless steel or some equivalent. The pins 38, 40 should be corrosion resistant, smooth and have a very hard surface having a hardness as measured by the Rockwell method, of greater than 60). Other materials include, but are not limited to, titanium nitride (TiN) coated titanium or TiN 27 coated stainless steel. Lower spindle support 28 and upper spindle support 29 are constructed of a shed-resistant material. Preferably, the shed-resistant material has other characteristics such as remaining dimensionally stable after steam sterilization or autoclaving, having a low coefficient of friction so as to produce minimum resistance, and/or being a medical grade Class VI polymeric material.

One such material is a specially formulated compound of virgin polytetrafluoroethylene and fillers (having Food and Drug Administration Master File Number MAF 288 and sold under the tradename RULON, available from S Furon Dixon, 386 Metacom Ave., Bristol, RI 02809). This material has been found to be particularly advantageous because as the lower spindle and upper spindle rotate within the spindle supports this material does not shed or leave undesired particles in the material as it passes over the spindle pins.

Conventional graphite bearings are typically unacceptable because they shed tiny black particles that discolor and/or contaminate the material being concentrated such that it can not be used for human injection.

In another embodiment, lower spindle pin 38 and upper spindle pin 40 are a shed-resistant material and the lower spindle support 28 and upper spindle support 29 are highly polished hard chrome plated 316 stainless steel or some equivalent. The supports 28, 29 should be corrosion resistant, smooth and have a very hard surface RF 60). Other materials include, but are not limited to, TiN coated titanium or TiN coated stainless steel.

In either embodiment, it is preferable to have the semisolid or gel flow over the spindles and spindle supports so that each of these components remain relatively cool, lubricated, and there is no concern of a seal failing and exposing the semisolid or gel to undesirable contamination or discoloration by a non-shed resistant bearing. Important aspects of the present invention are the shedresistant material used for the bearing interface, the ability to do aseptic 28 processing for producing a human injectable product, and the ability to process semisolids or gels without clogging the system.

Rotatable body 30 is rotated by any number of means known by those of ordinary skill in the art. In the embodiment shown in FIG. 2, a magnetic drive coupling is used to rotate the rotatable body. A motor or power source (not shown) rotates the ring of magnets 42 located around the base of apparatus Another magnet or set of magnets 44 are located in the base of rotatable body As the ring of magnets 42 is rotated, the magnetic forces act on magnet 44 to cause the rotatable body to rotate. Preferably, the rotatable body is rotated in a range of 500 to 4000 rpm, more preferably in a range of 1000 to 3000 rpm, and most preferably at 1500 to 2000 rpm for a rotatable body diameter of 4 inches and an annular gap of 3/16 inches. Vortex flow concentration principles scale up linearly in size, therefore one of ordinary skill in the art can determine the necessary dimensions of the elements of the apparatus for the particular desired processing rate using the equations discussed above.

As the rotatable body is rotated, pump 16 pushes the material through openings 22,23 adjacent to inlet 18, over bearing interface between lower spindle 34 and lower spindle support 28, and into annular gap 46 between outer side wall of rotatable body 30 and the inner side wall 52 of membrane (or filter) 48.

The material is separated at the interface with the membrane into a retentate that stays in annular gap 46 and a permeate, which is primarily water, soluble molecules, and small particles, that passes through the membrane. The permeate passes into annular space 54 located between outer side wall 56 of membrane 48 and inner side wall 58 of apparatus 10. The permeate can then be drained out of the apparatus through drains 60,61. There can be one or more drains and the drains can be located anywhere along the apparatus. In one embodiment, a slight positive pressure (approximately 2 to 4 psi) can be maintained on the permeate 29 side to prevent pulling the permeate through the membrane and prevent membrane fouling.

The Taylor vortices discussed above are created in annular gap 46. The vortices act to prevent the openings 62 in membrane 48 from becoming clogged, blinded, fouled, etc. If the vortices were not present, a gel layer would begin to develop on the inner side wall of the membrane and decrease the flux through the membrane. The vortices allow liquids and small particles to pass through the membrane while pulling the gel or larger particles back away from the membrane openings. Each vortex acts as a whirlpool pulling particles and macromolecules away from the membrane surface. The result is that the material between the rotatable body and the membrane becomes more concentrated forming a gel or semisolid while maintaining stable permeate flow rates. The gel or semisolid is circulated (or recirculated) through the apparatus over the bearing interface between upper spindle 36 and upper spindle support 29, through openings 22,33, and out through outlet 20. The gel or semisolid is recirculated through system 14 as many times as required until the desired concentration is achieved. In order to adjust the size of annular gap 46 between inner side wall 52 of membrane 48 and outer side wall 50 of rotatable body 30, membranes of different inside diameters can be used or rotatable bodies of different outside diameters can be used in the apparatus.

Lower flange member 64 is sized to create a tight friction fit with the bottom of apparatus 10 and, in one embodiment includes O-ring 70 to prevent leakage. In another embodiment, lower flange member 64 is integral with flange 66 around the base of apparatus 10. Membrane 48 is sized to create a tight friction fit with inner side wall 58 around the base of apparatus 10 and, in one embodiment includes O-ring 68 to prevent leakage. O-rings 72 and 74 can also be provided to prevent leakage around lower support 24. O-rings 76 and 78 can also be provided to prevent leakage around upper support 25. At all locations where O-rings are used, it is preferable to round off the edges of the 0-ring seat so that the O-ring is not pinched when the two parts are mated. The pinching of the 0-ring can result in parts of the O-ring skiving off and discoloring and/or contaminating the pharmaceutical or biological material. Preferably, the O-rings are made of medical grade elastomer.

In one embodiment, upper flange 80 around the top of membrane 48 is sized to fit between flange 82 around the top of apparatus 10 and upper flange member 84 with O-rings 86 and 88 provided to prevent leakage. Fasteners are used to create a tight fit between flange 82 and upper flange 80, and between gupper flange 80 and upper flange member 84. Fasteners such as shown in FIG. 2 are advantageous because the bolt is integrally attached to flange 82 and the nut simply has to be removed for disassembling, cleaning, sterilizing, servicing, etc.

the apparatus. As one of ordinary skill in the art will recognize other means can be used in place of fasteners 90, such as clamps, twist-locks, threaded fittings, etc. Likewise, upper flange 80 of membrane 48 does not have to extend between upper flange member 84 and flange 82. It can fit within the inner wall of the apparatus such that upper flange member 84 and flange 82 mate face to face as is shown at the base of the membrane in FIG. 2. Upper support 25 and outlet are attached to upper flange member 84 by fasteners 26. As will be recognized by one of ordinary skill in the art other fasteners can be used. Fasteners 26 are advantageous, however, because they are removable which allows for ease in cleaning and sterilization as well as repair of the inlet, apparatus, and support.

The apparatus has been described with respect to upper and lower components, however the apparatus is not limited to a vertical orientation.

Because of the forces involved during operation, the apparatus can be operated horizontally or on an incline as well. In addition, the rotatable body and the 31 membrane do not have to both be cylinders. One or both could be a cone for example.

As can best be seen in FIG. 5, lower support 24 (likewise, upper support has elongated openings 22 spaced angularly around the center opening. The elongated openings are particularly advantageous because they allow the semisolid or gel to pass through more easily than smaller, circular openings. The elongated openings prevent clogging, help to decrease pressure differentials and increase the flow rate. Other configurations of enlarged openings are beneficial as well. Inlet 18 and outlet 20 are of large inner diameter and straight without any bends or elbows for the same reasons and to aid also in aseptic assembly of the apparatus. Preferably, all of the parts previously described, except for the magnets, lower spindle support, upper spindle support and the O-rings, are made from 316L stainless steel (polished to a 32 RMS finish) for ease in cleaning and sterilization, or some equivalent.

15 Membrane or filter 48 can be made from many different materials and have various pore sizes. The membranes can be described as permeable or semipermeable. Stainless steel membranes ranging from those manufactured from sintered steel powder with a pore rating of 0.2pm up to those manufactured from sintered steel powder or steel fibers with 1, 3, 5, 10, 20, 50 and 100/m pore.

ratings can be used. Stainless steel screens with pore sizes of 20 to 200plm can also be used. The advantage of steel membranes is that they can be cleaned with a wide array of agents, including caustics, and they can withstand exposure to repeated steam sterilization cycles. Hydrophilic polymeric ultrafilters and microfilters made from polysulfone or a cross-linked polyacrylonitrile polymer in pore sizes ranging from 10kD to 0.2grm can also be used. The preferred membrane pore size is dependent on the particle or fiber size of the material to be concentrated. In the case of collagen 0.2pm to 5j/m is preferred, more be 0.: 0...C

C,

C.

C

preferably luim to 3Lm.

Experimental Experiments were conducted which demonstrated the feasibility of employing vortex flow filtration devices to concentrate collagen compositions. The filtration system used was that shown in FIGS. Specifically, the vortex flow filtration devices employed were a Membrex Mini-Pacesetter'" (Examples 1-2) and Membrex PaceSetter' (Example 3) both from Membrex, Inc. Fairfield, N.J. The membranes used were also available from Membrex, Inc. The devices were modified so that the lower spindle support and upper spindle support are constructed of RULON", as described above. The collagen compositions tested were prepared from a 0.30% bovine collagen solution (pH Collagen was caused to precipitate by the addition sufficient amounts of 0.2M sodium phosphate buffer solution (pH 11) to form a collagen suspension containing about 0.27% collagen at about pH 7. As shown in Examples 1, 2, and 3, this 0.27% collagen composition was concentrated to 8.5% and 7.6% collagen gels, respectively.

EXAMPLE 1 A collagen suspension comprising 0.27% collagen (about pH 7) and having the consistency of a thin, readily flowable slurry was concentrated by vortex flow filtration using a 400 cm 2 1im stainless steel membrane. Taylor vortices are created in the annular gap by the rotor (or rotatable body) spinning at 1500 rpm inside the cylindrical membrane. Product was recirculated through the system at approximately 2 LUmin and heat from the rotor and the pump was removed by a heat exchanger on the return to the reservoir feed tank. Permeate was collected through the membrane which retained the collagen suspension. The permeate flow rate was controlled by a peristaltic pump to maintain a low transmembrane pressure. A slight positive pressure, about 2-4 psi, was maintained on the permeate side of the

L

membrane to avoid pulling the permeate through the membrane and resultant membrane fouling. 39 kg of collagen suspension were concentrated from an 0.27% suspension to an 8.1% gel at a rate of 300 mL/min. The flux for the last 3 liters declined to about 150 mLlmin. Total concentration time was 2.5 hours. The final concentrate Was recovered from the system by pumping the gel and then blowing the residual out with nitrogen.

Alternatively the residual concentrate can be rinsed out with water or buffer solutions.

10 EXAMPLE 2 S. A collagen suspension comprising 0.27% collagen (about pH 7) and having the consistency of a thin, readily flowable slurry was concentrated by vortex flow filtration using a 400 cm 2 1Rm stainless steel membrane. Taylor vortices are created in the annular gap by the rotor (or rotatable body) 15 spinning at 1500 rpm inside the cylindrical membrane. Product was S. recirculated through the system at approximately 2 L/min and heat from the rotor and the pump was removed by a heat exchanger on the return to the reservoir feed tank. Permeate was collected through the membrane which retained the collagen suspension. The permeate flow rate was controlled by a peristaltic pump to maintain a low transmembrane pressure. A slight positive pressure, about 2 to 4 psi, was maintained on the permeate side of the membrane for the majority of the run to avoid pulling the permeate through the membrane and resultant membrane fouling. 82 kg of collagen suspension were concentrated from an 0.27% suspension to an 8.5% collagen gel at a rate of 0.3 L/min. Total concentration time was 4 hours and 39 minutes. The final concentrate was recovered from the system by displacing the gel with nitrogen at 15 psi pressure.

EXAMPLE 3 A collagen suspension comprising 0.27% collagen (about pH 7) and having the consistency of a thin, readily flowable slurry was concentrated by vortex flow filtration using a 2300 cm 2 ltm stainless steel membrane. Taylor vortices are created in the annular gap by the rotor (or rotatable body) spinning at 1800 rpm inside the cylindrical membrane. Product was recirculated through the system at approximately 11 Lmin and heat from the rotor and the pump was removed by a heat exchanger on the return to the reservoir feed tank. The permeate flow rate was controlled by a peristaltic 10 pump to maintain a low transmembrane pressure. A slight positive pressure, about 2 to 4 psi was maintained on the permeate side of the membrane for the majority of the run to avoid pulling the permeate through the membrane and resultant membrane fouling. 76 kg of collagen suspension were concentrated from an 0.27% suspension to a 7.6% gel at a rate of 1.3 L/min.

15 The flux for the last 6 liters declined to between 0.2 to 0.8 Imin. Total concentration time was 75 minutes. The final concentrate was recovered from the system by displacing the gel with air at 15 psi pressure.

i The foregoing has described the principles, preferred embodiments and modes of operation of the present invention. However, the invention should not be construed as being limited to the particular embodiments discussed.

For example, as already set out above, filtration systems using a membrane mounted on an inner body which rotates within a stationary body as disclosed in U.S. Patent Nos. 4,790,942, 4,876,013 and 4,911,847 issued to Shmidt et al., which are incorporated herein by reference in their entirety, are within the scope of the claimed invention. As are systems that use counter-rotating membranes and solid bodies or counter-rotating membranes alone. Thus, the above-described embodiments should be regarded as illustrative rather than restrictive, and it should be appreciated that variations may be made other than those discussed by workers of ordinary Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

o• *o*o* **o

Claims (36)

1. An apparatus having a permeable or semi-permeable membrane for aseptic vortex flow concentration of a dispersion, including: a rotatable body having an outer side wall, an upper spindle, and a lower spindle; an outer body having an upper end with openings therein, a lower end with openings therein, and an inner side wall spaced from the outer side wall of the rotatable body to define an annular space for receiving the permeable or semi-permeable membrane; an upper spindle support in the upper end of the outer body for receiving the upper spindle for producing a shed-resistant interface; a lower spindle support in the lower end of the outer body for receiving the lower spindle for producing a shed-resistant interface; and means for rotating the rotatable body about an axis through the upper spindle and the lower spindle at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotatable body and the permeable or semi-permeable membrane; said upper spindle support being located adjacent to the openings in the upper end and the lower spindle support being located adjacent to the openings in the lower end such that the dispersion passes through the openings and lubricates the upper spindle support and the lower spindle support.

2. The apparatus of claim 1 wherein said upper spindle and lower spindle are shed- resistant material. S

3. The apparatus of claim 1 or claim 2 wherein said upper spindle support and lower spindle support are shed-resistant material.

4. The apparatus of claim 3 wherein the shed-resistant upper spindle support and the shed-resistant lower spindle support are bushings which remain dimensionally stable after steam sterilization.

The apparatus of claim 3 or claim 4 wherein the shed-resistant upper spindle support and the shed-resistant lower spindle support are medical grade USP Class VI 35 material. 0.00°

6. The apparatus of any one of the preceding claims wherein the means for rotating the rotatable body is a magnetic drive system.

7. The apparatus of any one of the preceding claims further including: an exit port located in said outer body; and said openings in the lower end and the upper end of the outer body are in fluid communication with the annular gap between the outer side wall of the rotatable body and the permeable or semi-permeable membrane such that the dispersion enters the annular gap and the dispersion is concentrated in the annular gap as a permeate passes through the permeable or semi-permeable membrane and exits through the exit port.

8. The apparatus of any one of claims 1 to 6 wherein the dispersion enters the annular gap through the openings in the lower end and the concentrated dispersion exits through the openings in the upper end.

9. The apparatus of any one of the preceding claims wherein the openings in the lower end and the upper end are elongated to prevent clogging by the concentrated dispersion.

10. An apparatus having a filter for aseptic vortex flow concentration of a dispersion including: a housing having an upper end with an outlet therein, a lower end with an inlet therein, an inner side wall, an upper rotor support in the upper end of the housing, and a lower rotor support in the lower end of the housing; S 25 a rotor having an upper shed-resistant member and a lower shed-resistant S member rotatably received in the upper rotor support and the lower rotor support, the rotor having an outer side wall spaced from the inner side wall of the housing to define an annular space for receiving the filter; and means for rotating the rotor about an axis through the center of the rotor at a rate sufficient to produce Taylor vortices in an annular gap between the outer side wall of the rotor and the filter; said upper rotor support being located adjacent to the outlet in the upper end and 0*0* the lower rotor support being located adjacent to the inlet in the lower end such that the dispersion passes over the upper shed-resistant member and the lower shed-resistant member; an exit port located in said housing; and oooo an exit port located in said housing; and said inlet in the lower end and the outlet in the upper end of the housing are in fluid communication with the annular gap between the outer side wall of the rotor and the filter such that the dispersion enters the annular gap and the dispersion is concentrated in the annular gap as a permeate passes through the filter and exits through the exit port.

11. The apparatus of claim 10 wherein the upper shed-resistant member and lower shed-resistant member are spindles which remain dimensionally stable after steam sterilization.

12. The apparatus of claim 10 or claim 11 wherein the upper shed-resistant member and the lower shed-resistant member are medical grade USP Class VI material.

13. The apparatus of any one of claims 10 to 12 wherein the means for rotating the rotor is a magnetic drive system.

14. The apparatus of any one of claims 10 to 13 wherein the dispersion enters the annular gap through the inlet in the lower end and the concentrated dispersion exits through the outlet in the upper end.

The apparatus of any one of claims 10 to 14 wherein the inlet in the lower end and the outlet in the upper end have a plurality of elongated openings to prevent clogging. 25

16. A process for separating a collagen composition into a permeate portion and a concentrated retentate portion, which includes the steps of: providing a vortex flow filtration apparatus according to any one of claims 1 to 15 having an inlet and an outlet; causing the collagen composition to flow through the inlet.and into the V 30 gap; and rotating the rotor means at a speed effective to separate the collagen composition into permeate and concentrated retentate portions. 000o

17. The process of claim 16 further including the steps of withdrawing the permeate o# during rotation of the rotor means and withdrawing the concentrated retentate during rotation of the rotor means.

18. The process of claim 16 or claim 17 wherein the filter means is located on the inner surface of the outer member.

19. The process of claim 16 wherein the filter means is located on the outer surface of the inner member.

The process of claim 16 wherein the filter is mounted on the outer wall of the rotor member.

21. The process of any one of claims 16 to 20 wherein the rotor means is rotated at between about 1000 and about 3000 rpm.

22. The process of any one of claims 16 to 21 wherein the concentrated retentate includes about 0.25% to about 15% collagen.

23. The process of claim 22 wherein the concentrated retentate includes about 6% to about 8% collagen.

24. The process of any one of claims 16 to 23 wherein the retentate is recirculated into the inlet to produce a higher concentrated retentate.

25. The process of claim 24 wherein the concentrated retentate includes about 0.25% to about 15% collagen.

S26. The process of claim 24 wherein the concentrated retentate includes about 6% to about 8% collagen.

27. A process for producing a concentrated collagen composition from a collagen suspension, which includes the steps of: providing a vortex flow filtration apparatus according to any one of claims 1 to 15 having an inlet and an outlet; causing the collagen suspension to flow through the inlet and into the gap; rotating the rotor means at speeds effective to separate the collagen suspension into a permeate and a retentate; *000. 35 suspension into a permeate and a retentate; *0 0 recirculating at least a portion of the retentate into the fluid gap for further separation into permeate and retentate; and continuing steps and until the retentate has a collagen concentration of between about 3% to about 15%

28. The process of claim 27 further including the steps of withdrawing the permeate during rotation of the rotor means and withdrawing the concentrated retentate during rotation of the rotor means.

29. The process of claim 27 or claim 28 wherein the filter means is being located on the inner surface of the outer member.

The process of claim 27 or claim 28 wherein the filter means is being located on the outer surface of the inner member.

31. The process of claim 27 or claim 28 wherein the filter is mounted on the outer wall of the rotor means.

32. The process of any one of claims 27 to 31 wherein the rotor means is rotated at between about 1000 and about 3000 rpm.

33. The process of any one of claims 27 to 32 wherein the concentrated retentate includes about 0.25% to about 15% collagen.

34. The process of any one of claims 27 to 33 wherein the concentrated retentate includes about 6% to about 8% collagen.

The process of any one of claims 27 to 34 wherein the concentrated retentate includes about 6% to about 8% collagen. *00o oo00

36. A process for separating a collagen composition into a permeate portion and a concentrated retentate portion substantially as hereinbefore described with reference to any one of the examples. DATED this seveth day of July 2003 MATRiX PHARMACEUTICAL, INC., Patent Attorneys for the Applicant: F.B. RICE Co. S 0* 44 4 44 44 44 4 4 S S S S. S. 49 5 4 S. 55 9 4 S S .54. S S S 4 4.55