AU779126B2 - Separation of fibrinogen from plasma proteases - Google Patents

Description

WO 01/48016 PCT/AU00/01585 1 Separation of fibrinogen from plasma proteases FIELD OF THE INVENTION The present invention relates to methods for purifying fibrinogen. In one aspect, the present invention relates to a method of separating fibrinogen from plasma fraction I precipitate. In another aspect, the invention relates to the purification of fibrinogen using ion exchange chromatography.

BACKGROUND OF THE INVENTION The isolation of human fibrinogen has traditionally been carried out by classical plasma fractionation methods. Fibrinogen is precipitated from plasma either with ethanol (Blomback and Blomback, 1956), ammonium sulphate (Takeda, 1996), P alanine/glycine (Jakobsen and Kieruif, 1976), polymers (polyethelene glycol) and low ionic strength solutions (Holm, 1985) with relative high yield and homogeneity.

Further purification of fibrinogen precipitates can be achieved by ion-exchange chromatography conditions (Stathakis et al, 1978) and affinity chromatography (Kuyas et al, 1990). Specific contaminants can be absorbed out for example fibronectin on immobilised gelatine and plasminogen an immobilised lysine (Vuento et al, 1979).

Precipitation methods are widely used for the manufacture of commercial fibrinogen. Chromatographic methods are now being explored as an alternative or to improve the purity of fibrinogen concentrates.

WO 99/37680 describes a method for the large scale separation of fibrinogen from other blood proteins in human blood plasma. The process involves the use of a heparin precipitated paste as a starting material for the purification of fibrinogen. The heparin precipitated paste is a by-product from the manufacturing process of Factor VIII (Antihaemophilic Factor,

AHF).

Attempts to produce fibrinogen free of plasminogen or to purify plasminogen itself have been widely published in the literature. The most common method is to utilise the ability of lysine to bind to one of the two "kringles" in the plasminogen molecule. The use of affinity chromatography step was first disclosed in a paper published by Deutsch and Mertz in 1970.

Baxter International Inc. utilised this technology, which incorporated the use of lysine-sepharose material in a dedicated step to remove plasminogen from WO 01/48016 PCT/AU00/01585 2 their fibrinogen as disclosed in the patent WO 95/25748 for the large scale manufacture of a fibrinogen concentrate free of destabilising levels of plasminogen product. Other techniques published in the scientific literature again utilise the binding of either lysine or e-amino caproic acid. However, they are employed to alter the solubility of the plasminogen molecule.

Following the addition of lysine to a dilute fibrinogen solution, the subsequent solution is then precipitated in the presence of 7% ethanol.

Removal of plasminogen is stated at greater than 90% with a repeat of the step leading to total removal of the contaminant (Mosesson, 1962).

Precipitation methods are widely used for the manufacture of commercial fibrinogen, however, the work published by Mosesson (1962) relys on a dilute solution of fibrinogen which is not a practical process for implementation at a production scale.

The use of ion-exchange chromatography and e-amino caproic acid to bind and elute plasminogen independent of pH or ionic strength was disclosed in a patent (WO 94/00483) lodged in 1994 by Novo Nordisk A/S describing the purification of kringle containing proteins. This method chooses S-sepharose as the resin of choice. Also, a combination of gel filtration and ion-exchange chromatography has been utilised to purify plasminogen. (Robbins et al, 1965).

SUMMARY OF THE INVENTION The present inventors have now found that fibrinogen may be recovered in a purified form from a starting material consisting of Fraction I paste. The fibrinogen recovered in this process is free of destabilising levels of plasminogen and other proteases. Fibrinogen recovered in this manner also contains factor XIII, which is required to enhance the cross-linking of fibrin polymers in the production of fibrin glue. Furthermore, the yields of fibrinogen obtained by this process are unexpectedly higher than those obtained in methods which use alternative starting materials, such as heparin precipitated paste.

The present inventors have also developed an improved method for recovering fibrinogen from an ion-exchange column which involves the addition of at least one co-amino acid to the fibrinogen-containing material WO 01/48016 PCT/AU00/01585 3 applied to the column or to the solution used to wash the column prior to elution of the fibrinogen.

When used herein, the phrase 'Traction I precipitate" refers to frozen plasma which has been thawed and the cryoprecipitate removed by centrifugation. The resultant cryosupernatant is then mixed with ethanol to precipitate Fraction I.

Accordingly, in a first aspect the present invention provides a method of purifying fibrinogen, which method comprises extracting fibrinogen from a Fraction I precipitate by admixing the Fraction I precipitate with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises salt at a concentration of at least 0.1M and heparin at a concentration of at least 10 IU/ml.

In a preferred embodiment of the first aspect, the concentration of salt is at least 0.2M, more preferably at least 0.4M, more preferably about 0.8M.

In a further preferred embodiment of the first aspect, the extraction buffer comprises at least one salt selected from the group consisting of chloride, phosphate and acetate salts, and more preferably comprises NaCl.

In a further preferred embodiment of the first aspect, the extraction buffer also comprises Tri-sodium citrate at a concentration of about In a further preferred embodiment of the first aspect, the extraction buffer further comprises at least one o-amino acid. Preferably, the at least one w0-amino acid is present in the extraction buffer at a concentration of at least In a further preferred embodiment of the first aspect, the extraction buffer comprises antithrombin I (ATIII) at a concentration of at least about 1 ir/ml.

In a further preferred embodiment of the first aspect, the extraction buffer comprises Tri-sodium citrate at a concentration of about 20mM, NaCL at a concentration of about 0.8M, heparin at a concentration of about IU/ml and at least one o-amino acid at a concentration of about Preferably the extraction buffer has a pH of about 7.3.

In a further preferred embodiment of the first aspect, the extraction of fibrinogen is performed at about 37°C. Preferably, the extraction is performed for at least 60, more preferably at least 90 minutes.

In a further preferred embodiment of the first aspect, the method further comprises the step of incubating the extracted fibrinogen solution SWO 01/48016 PCT/AU00/01585 4 with aluminium hydroxide followed by centrifugation and removal of the precipitate.

In a further preferred embodiment of the first aspect, the method further comprises the step of precipitating the fibrinogen from the extracted fibrinogen solution by the addition of a glycine saline (Gly/NaC1) buffer.

Preferably, the Gly/NaC1 buffer comprises glycine at a concentration of around 2.1M, Na-citrate at a concentration of around 20mM, sodium chloride at a concentration of around 3.6M and CaC1, at a concentration of around 2.4mM.

In a further preferred embodiment of the first aspect, the method further comprises the step of resolubilising the fibrinogen precipitate in a buffer comprising NaCI at a concentration of around 100mM, CaCl, at a concentration of around 1.1M, Na-citrate at a concentration of around tris at a concentration of around 10mM and sucrose at a concentration of around 45mM, preferably with a pH of about 6.9.

In a further preferred embodiment of the first aspect, the method further comprises the steps of: applying the extracted fibrinogen solution to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

In a further preferred embodiment of the first aspect, the method further comprises washing the ion exchange matrix with a buffer comprising at least one co-amino acid prior to eluting the fibrinogen from the matrix.

Preferably the wash buffer comprises the at least one Co-amino acid at a concentration of at least In a further preferred embodiment, the wash buffer comprises tris at a concentration of about 50mM, (ii) at least one ao-amino acid at a concentration of about 20mM, and NaCI at a concentration of about Preferably, the buffer has a pH of about 8.0. Preferably, the buffer has a conductivity of about 11.1 mS/cm.

In a second aspect, the present invention provides a method of purifying fibrinogen, which method comprises: extracting fibrinogen from a Fraction I precipitate by admixing the Fraction I precipitate with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the WO 01/48016 PCT/AU00/01585 extraction buffer comprises salt at a concentration of at least 0.1M; precipitating the fibrinogen; and solubilising the fibrinogen in a solution comprising at least one oamino acid at a concentration of at least 100mM.

In a preferred embodiment of the second aspect, the concentration of salt in the extraction buffer is at least 0.2M, more preferably at least 0.4M, more preferably about 0.8M.

In a further preferred embodiment of the second aspect, the extraction buffer comprises at least one salt selected from the group consisting of chloride, phosphate and acetate salts, and more preferably comprises NaCl.

Preferably, the extraction buffer also comprises Tri-sodium citrate at a concentration of about In a preferred embodiment of the second aspect, the extraction buffer further comprises heparin at a concentration of at least 10 IU/ml, more preferably about In a further preferred embodiment of the second aspect, the extraction buffer further comprises at least one o-amino acid. Preferably, the at least one e-amino acid is present in the extraction buffer at a concentration of at least In a further preferred embodiment of the second aspect, the extraction buffer comprises Na-citrate at a concentration of about 20mM, NaCl at a concentration of about 0.8M and heparin at a concentration of about IU/ml. Preferably the extraction buffer has a pH of about 7.3.

In a further preferred embodiment of the second aspect, the fibrinogen is precipitated in step by the addition of a glycine saline (Gly/NaC1) buffer. Preferably, the Gly/NaCi buffer comprises glycine at a concentration of around 2.1M, Na-citrate at a concentration of around 20mM, sodium chloride at a concentration of around 3.6M and CaCl, at a concentration of around 2.4mM.

In a further preferred embodiment of the second aspect, the fibrinogen precipitate is solubilised in step using a buffer comprising NaCl at a concentration of around 100mM, CaCl, at a concentration of around 1.1M, Na-citrate at a concentration of around 10mM, tris at a concentration of around 10mM and sucrose at a concentration of around 45mM. Preferably, the buffer has a pH of about 6.9.

WO 01/48016 PCT/AU00/01585 8 In a further preferred embodiment of the second aspect, the method further comprises: applying the fibrinogen solution from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

In a preferred embodiment of the second aspect, the method further comprises washing the ion exchange matrix with a buffer comprising at least one c-amino acid prior to eluting the fibrinogen from the matrix. Preferably the wash buffer comprises the at least one ac-amino acid at a concentration of at least In a further preferred embodiment of the second aspect, the wash buffer comprises tris at a concentration of about 50mM, (ii) at least one oamino acid at a concentration of about 20mM, and NaCl at a concentration of about 90mM. Preferably, the buffer has a pH of about 8.0. Preferably, the buffer has a conductivity of about 11.1 mS/cm.

In a further preferred embodiment of the second aspect, the fibrinogen containing solution (preferably comprising the o-amino acid) is diluted such that the conductivity is below 10.5 mS/cm before it is applied to the ion exchange matrix.

In a further preferred embodiment of the second aspect, the fibrinogen is eluted from the matrix in a buffer comprising about 10 mM Tris, citrate, 45mM sucrose; and NaCl at a concentration of between 200mM to 1.OM, more preferably about 400mM. Preferably, the buffer has a pH of about In a third aspect the present invention provides a method of purifying fibrinogen, which method comprises: extracting fibrinogen from a fibrinogen containing material by admixing the material with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises at least one o-amino acid at a concentration of at least applying the extraction buffer from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

WO 01/48016 PCT/AU00/01585 7 In a preferred embodiment of the third aspect, the method further comprises washing the ion exchange matrix after step with a solution comprising at least one o-amino acid.

In a fourth aspect the present invention provides a method of purifying fibrinogen from a fibrinogen containing solution which method comprises: applying the solution to an ion exchange matrix, under conditions such that fibrinogen binds to the matrix; washing the ion exchange matrix with a solution comprising at least one e-amino acid; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

In a preferred embodiment of the fourth aspect, the method further comprises adding at least one o-amino acid to the solution before applying to the ion exchange matrix.

In the context of the third and fourth aspects of the present invention, the fibrinogen containing material may be any material derived from plasma which includes fibrinogen. Examples of such solutions include, but are not limited to, plasma (including anti-coagulated plasma), or plasma fractions.

In preferred embodiment, the material is a heparin precipitated paste, which is a by-product in the manufacturing process of Factor VII. The heparin precipitated paste may be solubilised with a salt solution to provide a fibrinogen preparation of high specific activity. A process for precipitating fibrinogen from a cryoprecipitate extract using heparin as described in Winkelman et al. 1989, the entire contents of which are incorporated herein by reference. Alternatively, the fibrinogen containing material is extracted from Fraction 1 precipitate, preferably in accordance with a method of the first or second aspects of the present invention.

In a further preferred embodiment of the third or fourth aspects, the eamino acid is present in the extraction buffer at a concentration of between 5-500mM, more preferably between 50-500mM, and more preferably around 100mM.

In a further preferred embodiment of the third or fourth aspects, the fibrinogen containing solution (preferably comprising the o-amino acid) is diluted such that the conductivity is below 10.5 mS/cm before it is applied to the ion exchange matrix.

8 In a further preferred embodiment of the third or fourth aspects, the buffer used to wash the ion exchange matrix comprises tris at a concentration of about 50 mM, (ii) a so-amino acid at a concentration of about 20mM, and NaCl at a concentration of about 90mM. Preferably, the buffer has a pH of about 8.0. Preferably, the buffer has a conductivity of about 11.1 mS/cm.

In a further preferred embodiment of the third or fourth aspects, the fibrinogen is eluted from the matrix in a buffer comprising about 10 mM Tris, 10 mM citrate, mM sucrose; and NaCI at a concentration of between 200 mM to 1.OM, more preferably about 400-500 mM. Preferably, the buffer has a pH of about In a preferred embodiment of the first, second, third or fourth aspects of the present invention, the(D-amino acid contains at least 4 carbon atoms in the carbon chain between the carboxylic acid and the co-amino group. The carbon chain may be linear or cyclic. Examples of suitable linear o-amino acids are 4-aminobutyric acid, aminopentoic acid, 6-aminohexanoic acid e-amino caproic acid (EACA)), 7aminoheptanoic acid, 8-aminooctanoic acid, and arginine. Examples of cyclic o-amino acids are trans-4-aminomethyl cyclohexane carboxylic acid (tranexamic acid) and paraaminomethyl benzoic acids. In a particularly preferred embodiment, the o-amino acid is EACA.

The invention also provides a method for purifying fibrinogen, which method comprises: extracting fibrinogen from Fraction 1 precipitate by admixing Fraction 1 precipitate with an extraction buffer such that fibrinogen is solubilized in the extraction buffer, wherein the extraction buffer comprises at least one co-amino acid at a concentration of at least 25 applying the extraction buffer from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and S(d) optionally recovering the fibrinogen from the eluate.

The invention also provides a method for purifying fibrinogen which method comprises: extracting fibrinogen from a fibrinogen containing material by admixing the material with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises at least one co-amino acid at a S" concentration of at least 35 applying the extraction buffer from step to an ion exchange matrix o under conditions such that fibrinogen binds to the matrix; 8a washing the ion exchange matrix after step with a solution comprising at least one co-amino acid; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

Ion exchange matrices are known in the art and any suitable matrix may be used in the present invention. A preferred matrix is the MacroPrep HQ Resin (BioRad, catalogue no. 156-0041). In a further preferred embodiment, the ion exchange matrix is loaded into a column.

It will be appreciated by those skilled in the art that the methods of the third and fourth aspects have the potential to provide an alternative to affinity chromatography for the large scale production of fibrinogen free of destabilising levels of plasminogen and other proteases. The methods in these aspects require only a single processing step using ion exchange chromatography for the isolation of fibrinogen free of destabilising levels ofplasminogen and other proteases from biological fluids with a high recovery rate (approximately The use of this novel method for the purification of fibrinogen from blood proteins has the potential to enable a simpler method of manufacture leading to a product which is superior in both purity and stability.

S...i *o *ooo* o *oo° WO 01/48016 PCT/AU00/01585 9 The technology of the current invention offers many advantages with regards to both the manufacture of fibrinogen and the use of fibrinogen in a fibrin sealant product. The removal of plasminogen from the fibrinogen component allows the manufacturer the liberty of not having to add inhibitory agents, either human, animal or synthetically derived, in order to obtain the desired stability of the fibrinogen component and fibrin glue.

Addition of inhibitory agents can lend itself to other problems which are avoided by the removing plasminogen from the final product.

Finally, the production costs of an ion-exchange resin is far more economical than the cost of lysine-sepharose or immobilised lysine resin which are used in affinity chromatography procedures.

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.

I WO 01/48016 ~VOO1J8O16PCT/AUOO/01585 Abbreviations used herein:

TP

cP FXU1I

FII

Plasm.

FN

ATM

FIP

SFP

ASFP

GASFP

SDS-PAGE

GlyINaCl

SD

e-ACA

TNBP

Al(OH) 3

RT

PET

TEX

SHP

Total Protein Glottable protein Factor XIII Factor HI Plasminogen Fibronectin Antithrombin III Fraction 1 paste Solubilised fraction 1 paste Athydrogel absorbed solubilised fraction 1 paste Resolubilised Gly/NaCI precipitated solubilised fraction 1 paste Sodium dodecyl sulphate polyacrylarnide gel electrophoresis Glycine/Saline Solvent/detergent epsilon aiinocaproic acid Tri -N-butyl phosphate Aluminium hydroxide Room temperature Plasma Engineering Technology Ion exchange Solubilised heparin paste supernatant WO 01/48016 PCT/AU00/01585 11 BRIEF DESCRIPTION OF THE FIGURES Figure 1: Yield of clottable fibrinogen obtained in paste to buffer ratio study (I) Figure 2: Yield of total fibrinogen obtained in paste to buffer ratio study (1) Figure 3: Yield of total fibrinogen obtained in paste to buffer ratio study (2) Figure 4: Yield of clottable fibrinogen obtained over time during extraction of Fraction I paste.

Figure 5: Effect of temperature on extraction of fibrinogen from Fraction I paste.

Figure 6: Flow chart depicting a preferred fibrinogen purification process incorporating the ion-exchange chromatography method of the present invention.

WO 01/48016 PCT/AU00/01585 12 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1: Extraction of Fibrinogen from fraction 1 precipitate 1.1 Materials and Methods 1.1.1 Heparin paste extraction procedure Fraction I paste is extracted at a ratio of lg:8.33g heparin paste extraction buffer unless stated otherwise. The extraction is performed at room temperature for 2 hours.

1.1.2 Heparin paste extraction buffer 0.4 M NaC1, 5mM eACA, Na-citrate, pH 7.3.

1.1.3 Alhydrogel Absorption A solution of 2% aluminium hydroxide Al(OH) 3 also known as alhydrogel, is added to solubilised heparin paste superntant (SHP) at a concentration of 10.8%. The mixture is incubated with stirring for minutes at room temperature, centrifuged for 10 minutes, and the pellet discarded.

1.1.4 Gly/NaCI Precipitation The alhydrogel supernatant (ASFP) and Gly/NaCl buffer are heated to 3°C. The supernatant is then added to the Gly/NaCi buffer, over minutes, at a ratio of 1:2.05. The supernatant is then incubated at 30°C with stirring for 20 mins, before centrifuging for 10 mins at 5010g. The supernatant is discarded and the precipitate resolubilised using a volume of Buffer D equal to one third of the mass of the supernatant obtained after extraction of fibrinogen from Fraction I paste. The precipitate may be stirred at room temperature during resolubilisation.

W 01/48016 PCT/AU00/01585 13 1.1.5 Gly/NaCl buffer 2.1M glycine Na-citrate 3.6M NaC1 2.4mM CaCI.

1.1.6 Buffer D 100mM NaCl 1. mM CaC1l 10mM Na-citrate tris sucrose pH6.9 1.1.7 Solvent Detergent Treatment Solvent detergent treatment is performed by adding 1% polysorbate and 0.3% TNBP to the resolubilised Gly/NaCI precipitate (GASFP).

1.1.8 Wet Heat Treatment Solvent detergent treated fibrinogen is diluted 1/15 with concentrated sucrose/glycine buffer to a final concentration of approximately 1 mg/mL protein, 60% sucrose and 1 M glycine. The formulated product is heated to and incubated for 10 hours.

1.1.9 Ion Exchange Chromatography Wet heat treated fibrinogen is applied to an equilibrated anion exchange resin. After washing of the resin, the product is eluted using a saltcontaining buffer.

1.1.10 Stability at 37C In process samples were incubated at 37*C in a water bath and samples taken and frozen at regular time intervals. The stability samples were analysed by SDS-PAGE under reducing conditions. Stability was assessed qualitatively as the last time point where no degradation of the a subunit of fibrinogen was observable by eye on the gel.

WO 01/48016 PCT/AU00/01585 14 1.1.11 Extraction 1 Procedure Fraction 1 paste was obtained fresh from production. 6 g was extracted immediately using heparin paste extraction buffer. The solubilised fraction 1 paste was then aliquoted and stored frozen at -80 0 C until assayed.

1.1.12 Extraction 2 Procedure Fraction 1 paste was obtained fresh from production. 12 g was extracted immediately using heparin paste extraction buffer. The paste was treated with Al(OH), and then precipitated using Gly/NaC1 buffer. The precipitate was resolubilised in Buffer D. Samples were taken at each stage and frozen at -80°C until assayed. Remaining fraction 1 paste was stored at 0

C.

1.1.13 Extraction 3 (frozen paste) Procedure Fraction 1 paste (30g) was thawed at 370C and extracted using heparin paste extraction buffer. The solubilised fraction 1 paste was treated with AI(OH), and precipitated using Gly/NaCl buffer. The Gly/NaCl precipitate was then resolubilised using Buffer D. eACA was spiked into samples of the resolubilised Gly/NaC1 precipitate at concentrations of OmM, 20mM, 100mM, 200mM and 500mM. The samples were then assessed for stability at 37 0

C.

Resolubilised Gly/NaC1 precipitate was treated with SD and applied to the MacroPrep HQ ion exchange column. Fractions were collected and also assessed for stability at 37 0

C.

1.1.14 Extraction 4 Procedure Fraction 1 paste was obtained fresh from production. 40 g was extracted immediately using heparin paste extraction buffer. After 2 hours of extraction, the fraction 1 paste was not completely solubilised. The material was centrifuged* and the supernatant (solubilised fraction 1 paste #1) treated with Al(OH) 3 and Gly/NaCI precipitated. The Gly/NaCI precipitate was resolubilised using Buffer D. eACA was spiked into 2 sets of samples of the resolubilised Gly/NaCI precipitate at concentrations of OmM, 125mM, 250mM and 500mM. One group of samples was incubated at 37°C immediately and assessed for stability over time. The other group of samples was stored frozen at -80 0 C for 60 hours, thawed and then incubated at 37 0

C

WO 01/48016 PCT/AU00/01585 for stability. The remaining resolubilised Gly/NaCl precipitate was SD treated and applied to the ion exchange column.

*The non-solubilised fraction 1 material (14.13 g) was then re-extracted in heparin paste extraction buffer containing 0.8 M NaC1. The solubilised fraction 1 material was aliquoted and stored frozen at -80 0

C.

1.1.15 Addition of ATHI to extraction buffer Fraction 1 paste was obtained from production and half was stored for 4.5 days at 4 0 C and the other half at -80 0 C. In this experiment, extractions were performed using the 4 0 C (fresh paste) and the -80 0 C (frozen paste) extracted in buffer with and without 1 IU/mL ATII. An additional change to the standard extraction buffer was the increase in salt concentration to 0.8 M.

Fraction 1 paste (6g) was extracted under each of the following conditions: Fresh paste extracted in 20mM NaCitrate, 5mM sACA, 0.8M NaC1, pH 7.3.

Fresh paste extracted in 20mM NaCitrate, 5mM sACA, 0.8M NaC1, 1 IU/mL ATII, pH 7.3.

Frozen paste thawed at 37 0 C and then extracted in NaCitrate, 5mM sACA, 0.8M NaCl, pH 7.3.

Frozen paste thawed at 37 0 C and then extracted in NaCitrate, 5mM sACA, 0.8M NaCl, 1 IU/mL ATIII, pH 7.3.

The solubilised fraction 1 paste was subjected to alhydrogel absorption and precipitated using Gly/NaCl buffer. The precipitates were then split in half. Half the precipitate was stored at -80 0 C and the other half was resolubilised using Buffer D. The resolubilised precipitate was then split in half again and 0 sACA or 250mM sACA was added. The samples were then assessed for stability at 37 0

C.

The frozen Gly/NaCl ppt was thawed at 370C and resolubilised using Buffer D 100 mM 6ACA (warmed to 30 0 Resolubilisation was performed at 300C. The samples were then assessed for stability at 37 0

C.

WO 01/48016 PCT/AU00/01585 16 1.1.16 Addition of heparin to extraction buffer Fraction 1 paste was obtained from production after storage for 3 days at 4°C. In this experiment, 4 extractions were performed using buffer with and without 1 IU/mL ATI in the presence of 20 IU/mL or 60 IU/mL heparin.

Fraction 1 paste (6g) was extracted under each of the following conditions: Fresh paste extracted in 20 mM NaCitrate, 5 mM sACA, 0.4 M NaCI, 20 IU/mL heparin pH 7.3.

Fresh paste extracted in 20 mM NaCitrate, 5 mM sACA, 0.4 M NaC1, 60 IU/mL heparin, pH 7.3.

Fresh paste extracted in 20 mM NaCitrate, 5 mM sACA, 0.4 M NaC1, 20 IU/mL heparin, 1 lU/mL ATm, pH 7.3.

Fresh paste extracted in 20 mM NaCitrate, 5 mM sACA, 0.8 M NaCI, 60 IU/mL heparin, 1 IU/mL ATI, pH 7.3.

The solubilised fraction 1 paste was treated with Al(OH) 3 and precipitated using Gly/NaC1 buffer. The precipitates were then split in half.

Half the precipitate was stored at -80 0 C and the other half was resolubilised using Buffer D. The resolubilised precipitate was then split in half again and 0 sACA or 250 mM sACA was added. The samples were then assessed for stability at 37 0

C.

Resolubilisation of the frozen pellet was performed by the addition of Buffer D 100 mM EACA (warmed to 30 0 C) into the frozen Gly/NaCl precipitates. Resolubilisation was performed at 30 0 C. The samples were then assessed for stability at 37 0

C.

1.1.17 Paste:Buffer ratio study Study I: Fraction 1 paste was obtained fresh from production. 1.5 g, 3 g, 4.5 g, 6 g, 7.5 g 9 g were resolubilised in 50 g of extraction buffer containing 0.8 M NaCl. Samples were taken for total and clottable protein. Remaining material was discarded.

Study II: Fraction 1 paste (Batch 3715001253) was obtained fresh from production. 4.5 g, 9 g, 13.5 g, 18 g, 22.5 g 27 g were resolubilised in 150 mL of extraction buffer containing 0.8 M NaC1, 60 IU/mL heparin at 37 0 C for minutes. Samples were taken for total and clottable protein.

WO 01/48016 PCT/AU00/01585 17 1.1.18 Extraction temperature study Fraction 1 paste was obtained fresh from production. 18 g was extracted in 150 mL of 20 mM NaCitrate, 5 mM eACA, 0.8 M NaC1, pH 7.3 for 2 hours at room temperature. Another 18 g was extracted in 150 mL of mM NaCitrate, 5 mM eACA, 0.8 M NaCl, pH 7.3 for 2 hours at 37 0 C. Samples were taken at 30 min., 60 min., 90 min. and 120 min. throughout the extraction for total and clottable protein. The solubilised paste was then centrifuged and another sample taken.

1.1.19 Production scale extraction study Production scale extraction I: Fraction 1 paste was obtained fresh from production. 20.0 kg was extracted by PET group in 20 mM NaCitrate, 5 mM sACA, 0.8 M NaCI, IU/mL heparin, pH 7.3 at a ratio of 1 g paste 8.33 g buffer. Extraction was performed at 37 0 c for 90 min.

Solubilised fraction 1 paste was then subjected to alhydrogel absorption and Gly/NaCl precipitation were performed. The precipitate was split in two and half the precipitate resolubilised in Buffer D containing 100 mM eACA and the other half frozen at -80 0 C. The resolubilised Gly/NaCl precipitate was then treated with SD, wet heat treated and applied to the ion exchange column. The eluate was collected, sampled and frozen at -80 0

C.

Resolubilisation of the frozen pellet was performed by the addition of Buffer D 100 mM sACA (warmed to 30 0 C) into the frozen Gly/NaCI precipitates. Resolubilisation was performed at 30 0 C. The samples were then assessed for stability at 37 0

C.

Production scale extraction I: Fraction 1 paste was obtained fresh from production. 30.0 kg was extracted by PET group in 20 mM NaCitrate, 5 mM eACA, 0.8 M NaC1, IU/mL heparin, pH 7.3 at a ratio of 1 g paste 8.33 g buffer. Extraction was performed at 37 0 C for 90 min. Samples were taken for total and clottable protein.

WO 01/48016 PCT/AU00/01585 18 1.2 Results 1.2.1 Extraction 1 Procedure Fraction 1 paste (6g) was solubilised in 50 mL extraction buffer.

Following centrifugation, 52.47 g supernatant was collected and the pellet discarded.

Protein characterisation The solubilised fraction 1 paste was assayed for total protein, clottable protein, factor XIII, plasminogen and fibronectin as detailed in Table 1. The yield of fibrinogen per kilogram of plasma is calculated in Table 2. Size exclusion analysis was performed using Superose 6, the results of which are detailed in Table 3.

Table 1 Protein characterisation of solubilised fraction 1 paste Sample SFP Protein Fibrinogen Total %CP FXIII Plasminogen FN (mg/mL) (mg/mL) fibrinogen (IU/mL) (g/mL) (mg/mL) SFP 52.7 26.56 17.20 902.5 65 13.57 125-129 0.44 The characterisation of the solubilised fraction 1 paste shows that high levels of protein are extracted of which approximately 65% is clottable protein or fibrinogen. The solubilised fraction 1 paste also contains high levels of factor XIII and plasminogen but has low levels of fibronectin.

WO 01/48016 PCT/AU00/01585 19 Table 2 Yield of fibrinogen from solubilised fraction 1 paste Sample Total F1 F 1 paste Total Mass of Fibrinogen YIELD paste extracted flbrinogen starting plasma (g/kg plasma) generated (mg) (kg) SFP 60600 6.02 902.5 7476 1.22 The yield of fibrinogen from solubilised fraction 1 paste is high in comparison to solubilised heparin paste, with 1.22 g fibrinogen extracted per kilogram of plasma.

Table 3 Superose 6 analysis ofsolubilsed fraction 1 paste Sample Area Area Area Area other aggregate 1 aggregate 2 fibrinogen LMW proteins SFP 1.24 4.89 65.88 28.0 Superose 6 analysis of solubilised fraction 1 paste shows approximately 65% fibrinogen monomer with low levels of aggregates but the presence of low molecular weight proteins.

SDS-PAGE analysis SDS-PAGE analysis results show the presence of high molecular weight proteins under non-reducing conditions and three major bands at approximately 40-60 kDa under reducing conditions. This profile is typical of material rich in fibrinogen.

Stability at 37C The stability of solubilised fraction 1 paste was approximately 24 hours. The solubilised fraction 1 paste sample clotted between 24 hrs and 32 hrs incubation at 37 0

C.

WO 01/48016 PCT/AU00/01585 1.2.2 Extraction 2 Procedure Fraction 1 paste (12.06g) was extracted in 100.5 mL extraction buffer, centrifuged, Al(OH) 3 absorbed and Gly/NaCi precipitated.

Protein Characterisation All samples were assayed for total protein, clottable protein, factor XIII, factor II, plasminogen and fibronectin and the yield of fibrinogen per kilogram of plasma calculated (Table Size exclusion analysis was performed using Superose 6, the results of which are detailed in Table Table 4 Characterisation summary Sample Protein Fibrinogen Total CP FXIII II Plasm FN Fibrinogen mg/mL mg/mL fibrinogen IU/mL IU/mL pg/mL mg/mL YIELD (mg) (g/kg plasma) SFP 28.11 20.42 2200 73 12.61 0.53 120.35 0.64 1.83 ASFP 19.43 15.00 1706 77 10.29 UD# 100.56 0.38 1.42 GASFP 17.63 16.26 1355 92 14.98 UD# 72.15 0.08 1.13 undetected Characterisation of solubilised fraction 1 paste showed extraction of high levels of protein of which approximately 73% was clottable protein.

This result is consistent with that obtained from Extraction I. Again, high levels of factor XIII and plasminogen and low levels of fibronectin were also extracted. The yield of fibrinogen per kilogram of plasma was also high at 1.83 g/kg.

Following Al(OH), absorption, factor II, which was observed to be 0.53 IU/mL in the solubilised fraction 1 paste, was undetectable. Clottable protein was observed to be 77% and the concentrations of FXIII, plasminogen and fibronectin were relatively unchanged. The yield of fibrinogen per kilogram of plasma decreased by approximately 22% which is an expected result over this step.

WO 01/48016 PCT/AU00/01585 21 Gly/NaCi precipitation increased the purity of the fibrinogen to 92% clottable and removed fibronectin to negligible levels.

Table Superose 6 analysis Sample %Area Area Area Area other LMW aggregate 1 aggregate 2 fibrinogen proteins SFP 10.85 4.78 52.47 31.90 ASFP 6.32 3.20 58.86 31.62 GASFP 8.25 10.59 74.24 6.93 Superose 6 analysis of in process samples showed the purification of fibrinogen over the Gly/NaC1 precipitation step with an increase in the fibrinogen peak from 59% to 74% of the total area.

SDS-PAGE analysis SDS-PAGE analysis of solubilised fraction 1 paste showed very similar protein composition to that generated in Extraction 1. SDS-PAGE analysis of in process samples also demonstrated the purification of fibrinogen over the Gly/NaCl step. The resolubilised Gly/NaCl precipitate sample contains fewer high molecular weight protein bands when analysed under reducing conditions and the absence of bands at 200 kDa, 150 kDa, and 55 kDa when analysed under non-reducing conditions.

1.2.3 Extraction 3 (frozen paste) Procedure Fraction 1 paste (21.13g) was extracted in 176 mL extraction buffer, centrifuged, AI(OH), absorbed and Gly/NaCl precipitated. On addition of product to Gly/NaC1 buffer, some product clotted. Resolubilised Gly/NaCi precipitate was SD treated, applied to an ion exchange column and the fibrinogen was eluted in a salt-containing buffer.

Protein Characterisation All samples were assayed for total protein, clottable protein, factor XIII, and factor II and the yield of fibrinogen per kilogram of plasma SWO 01/48016 PCT/AU00/01585 22 calculated (Table Size exclusion analysis was performed using Superose 6, the results of which are detailed in Table 7.

Table 6 Characterisation summary Sample Protein Fibrinogen Total Factor Factor Fibrinogen mg/mL mg/mL fibrinogen CP XII II g/Kg plasma rU/mL IU/mL SFP clotted clotted clotted clotted ASFP 17.79 10.49 2029 59 6.95 UD 0.96 GASFP 12.21 10.07 836 82 clotted UD 0.40 IEX 3.72 2.1 358 56 0.17 column eluate #undetected Characterisation of solubilised fraction 1 paste was not performed as the samples clotted on thawing. One sample of resolubilised Gly/NaCl precipitate also clotted on thawing and as a result no data is available for factor XIII levels at this step.

Samples that were analysed demonstrated similar profiles to the previous extraction experiments. Clottable protein was approximately after AI(OH), absorption and increased to greater than 80% after Gly/NaCl precipitation. Factor XIII was present at 7 IU/mL after Al(OH) 3 absorption and factor II was undetectable. The yield of fibrinogen per kilogram of plasma was low with only 0.4 g/kg detected after Gly/NaCl precipitation. The GASFP was then applied to the ion exchange column to purify fibrinogen from plasminogen.

WO 01/48016 PCT/AUOO/01585 23 Table 7 Superose 6 analysis Sample Area Area Area Area other aggregate 1 aggregate 2 fibrinogen fragments SFP clotted clotted clotted clotted ASFP 8.35 6.49 41.31 43.85 GASFP 6.42 19.59 63.56 10.42 Superose 6 analysis showed very high levels of low molecular weight proteins at the Al(OH), stage. Purification of fibrinogen was again seen after the Gly/NaC1 precipitation with an increase in the fibrinogen content from to 60% of the total area.

SDS-PAGE analysis The first sample of solubilised fraction 1 paste was not analysed by SDS-PAGE as the sample clotted on thawing. SDS-PAGE analysis of samples after Al(OH), and Gly/NaC1 steps, under reducing and non-reducing conditions, shows the purification of fibrinogen. Analysis of the ion exchange column eluate showed that the major protein component is fibrinogen.

Stability at 37°C The first sample (24hrs) of solubilised fraction 1 paste was not analysed by SDS-PAGE as the sample clotted on thawing. The remainder of the solubilised fraction 1 paste stability sample clotted somewhere between 24 hrs and 32 hrs after being left at 37 0 C. Analysis of the Al(OH) 3 sample showed evidence of fibrinogen breakdown at 24 hours.

After Gly/NaCI precipitation, fibrinogen was stable at 44 hours but breakdown was evident at 144 hours (no 72 hour sample was found). When 200 or 500 mM eACA was added to the resolubilised Gly/NaCl precipitate, fibrinogen was stable for greater than 240 hours.

After elution from the ion exchange column, fibrinogen was stable for at least 208 hours (the last time point tested) without the addition of any eACA.

WO 01/48016 PCT/AU00/01585 24 1.2.4 Extraction 4 Procedure Fresh fraction 1 paste (40.0g) was extracted in 333 mL extraction buffer, centrifuged, AI(OH) 3 absorbed and Gly/NaCl precipitated. ACA was spiked into 2 sets of samples of the resolubilised Gly/NaC1 precipitate at concentrations of 0 mM, 20 mM, 125 mM, 250 mM and 500 mM. One group of samples was incubated at 37 0 C immediately and assessed for stability over time. The other group of samples was stored frozen at -80 0 C for 60 hours, thawed and then incubated at 370C for stability. Resolubilised Gly/NaCI precipitate was SD treated, applied to an ion exchange column and the fibrinogen was eluted in a salt-containing buffer.

Protein Characterisation All samples were assayed for total protein, clottable protein, factor XII, factor I, and fibronectin and the yield of fibrinogen per kilogram of plasma calculated in Table 8. The pellet remaining after the first extraction was re-extracted with buffer containing 0.8 M NaC1. Protein characterisation of this sample and yield per kg plasma is detailed in Table 9. Superose 6 analysis was not performed.

Table 8 Characterisation summary Sample Protein Fibrinoge Total FXII FII FN Fibrinogen mg/mL n mg/mL fibrinogen CP IU/mL IU/mL mg/mL YIELD (g/kg plasma) SFP 23.69 16.09 5680 68 11.9 0.36 0.45 1.85 ASFP 17.39 11.68 4472 67 9.0 UD# 0.31 1.45 GASFP 19.82 17.58 4126 89 15.0 UD# 0.06 1.35 IEX Eluate 8.49 8.00 704 94 NT* NT* NT* 1.07 undetected not tested WO 01/48016 PCT/AU00/01585 Characterisation of in process samples showed results consistent with previous extractions of fresh fraction 1 paste. Approximately 68% clottable protein was extracted from the fraction 1 paste. AI(OH), treatment reduced factor II to undetectable levels and the Gly/NaCI precipitation increased clottable protein to 89% and reduced fibronectin to negligible levels. The yield of fibrinogen per kg plasma at the solubilised fraction 1 paste stage was 1.85 and dropped to 1.35 after AI(OH), treatment and Gly/NaCl precipitation, which is expected over these steps. The recovery over the ion exchange chromatography step was approximately 76%.

Frozen GASFP was thawed and applied to the ion exchange column.

The eluate was shown to be high in clottable protein and contained low levels of plasminogen. The level of plasminogen in GASFP was not tested therefore the recovery of plasminogen over the ion exchange step cannot be calculated. However, 8 mg/mL fibrinogen and <0.2 tg/mL plasminogen in the eluate equates to less than 1.6 /ig/mL in a concentrated product of mg/mL. This result suggests that the ion exchange column is acting efficiently to remove plasminogen from the product 14.13 g fraction 1 paste (remaining after extraction 1) was solubilised in 117.7 mL fibrinogen extraction buffer (0.8 M NaC1, pH 7.3).

Table 9 Characterisation summary of solubilised fraction 1 paste #2 Sample Protein Fibrinogen Total Factor XIII Factor II FN Fibrinogen mg/mL mg/mL fibrinogen CP IU/mL IU/mL (mg/mL) YIELD (mg) (g_/kg plasma) SFP 26.18 17.73 2144 68 12.6 0.34 0.41 0.69 Therefore the total yield of fibrinogen per kg of plasma could be calculated as 1.85 0.7 g 2.5 g fibrinogen per kg of plasma at the solubilised fraction 1 paste stage.

SDS-PAGE analysis SDS-PAGE analysis shows the presence of fibrinogen in all fractions.

After Gly/NaC1 treatment, fewer protein bands are observed under reducing WO 01/48016 PCT/AU00/01585 26 conditions demonstrating the purification of the fibrinogen molecule that is also seen by protein characterisation.

Stability at 37°C The resolubilised Gly/NaCI precipitate is stable for 64 hours when zero and 20 mM ACA is added to the sample. After 64 hours the sample clotted.

With the addition of 125 mM eACA the sample is stable for 72 hours but breakdown of the molecule is evident at the 100 hr time point.

Addition of 250 mM and 500 mM eACA increases the stability of the resolubilised Gly/NaC1 precipitate to greater than 124 hrs, the last sample taken.

Resolubilised Gly/NaC1 samples spiked with zero or 20 mM sACA and frozen for 60 hours at -80°C before starting the stability trial, were observed to be less stable than resolubilised non-frozen precipitate. With the addition of zero or 20 mM SACA the samples were stable for 24 hrs after which time they clotted compared to 64 hours in the non-frozen samples. However, the addition of 125 mM, 250 mM and 500 mM eACA increases the stability of the fibrinogen to >96 hrs, the last time point taken, which did not differ significantly from 124 hours seen with the non-frozen sample. Thus, fibrinogen is stable at -80 0 C only with the addition of at least 125 mM eACA.

Stability analysis of the ion exchange eluate was shown to be >170 hrs without the addition of sACA.

1.2.5 Addtion of ATIl to extraction buffer Fraction 1 paste fresh and frozen, was extracted in 50 mL of the extraction buffer IU/mL ATIII), centrifuged, Al(OH), absorbed and Gly/NaC1 precipitated. The precipitate was split in half. Half the precipitate was stored at -800C and the other half was resolubilised using Buffer D. The resolubilised precipitate was split in half again and 0 SACA or 250 mM sACA was added. The samples were then assessed for stability at 37 0

C.

Protein Characterisation Extraction 1 Fresh fraction 1 paste (6.04g) was extracted in 50.34 g buffer containing 20 mM NaCitrate, 5 mM sACA, 0.8 M NaCI, pH 7.3. The WO 01/48016 PCT/AU00/01585 27 solubilised fraction 1 paste was then treated with Al(OH) 3 and precipitated using Gly/NaC1 buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma was calculated (Table Table Characterisation summary Sample Protein Fibrinogen Total fibrinogen clottable Fibrinogen mg/mL mg/mL (mg) protein Kg plasma SFP 19.56 12.14 608 62 0.85 GASFP 15.15 13.20 337 87 0.47 GASFP 13.89 11.84 313 85 0.44 250 mM eACA Characterisation of in process samples for total and clottable protein was again consistent with previous results. Approximately 60% clottable protein was extracted from fraction 1 paste which was increased to clottable protein after the Gly/NaCI precipitation step. The yield was lower than previous extractions with 0.85 g fibrinogen extracted per kilogram of plasma.

Extraction 2 Fresh fraction 1 paste (5.98g) was extracted in 49.84 g buffer containing 20 mM NaCitrate, 5 mM eACA, 0.8 M NaC1, 1 IU/ml ATIII pH 7.3.

The solubilised fraction 1 paste was then treated with AI(OH) 3 and precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table 11).

WO 01148016 PCT/AU00/01585 28 Table 11 Characterisation summary of solubilised Fraction 1 paste Sample Protein Fibrinogen Total clottable Fibrinogen mg/mL mg/mL fibrinogen protein g(Kg plasma SFP 16.15 10.48 524 65 0.74 GASFP 13.63 11.79 283 86.5 0.40 GASFP 250 mM 13.76 11.86 294 86 0.42 eACA Characterisation of in process samples generated using extraction buffer containing 1 IU/mL ATII was very similar to extraction buffer without ATIII. Approximately 6590 clottable protein was extracted from fraction 1 paste which was increased to 85% clottable protein after the Gly/NaCl precipitation step. The yield was also lower than previous extractions with 0.74 g fibrinogen extracted per kilogram of plasma.

Extraction 3 Frozen fraction 1 paste (6.06g) was thawed and extracted in 50.5 g buffer containing 20 mM NaCitrate, 5 mM 8ACA, 0.8 M NaCI, pH 7.3. The solubilised fraction 1 paste was then treated with AI(OH) 3 and precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table 12).

WO 01/48016 ~VO 0148016PCT/AUOO/01585 29 Table 12 Characterisation summary of solubilised Fraction 1 paste Sample Protein Fibrinogen Total fibrinogen 9/ clottable Fibrinogen mg/mL (mg) protein g/Kg plasma SFP 12.75 8.59 416 67 0.59 GASFP 7.84 6.57 140 84 0.20 GASFPI 250 mM 8.59 6.32 140 74 0.20 cAGA__ Characterisation of in process samples generated following extraction of frozen fraction 1 paste showed extraction of 67% clottable protein which increased to 84% following Gly/NaCl precipitation. These clottable protein results are consistent between fresh and frozen paste starting material. The yield per ilogram of plasma was lower than that extracted from fresh paste with less than 0.6 g fibrinogen extracted per kilogram of plasma.

Extraction 4 Frozen fraction 1 paste (6.07g) was thawed and extracted in 50.59 g buffer containing 20 mM NaCitrate, 5 mM eACA, 0.8 M NaCI, 1 ITJ/mL ATmJ, pH 7.3. The solubilised fraction 1 paste was treated with AI(OH), and precipitated using Gly/NaCI buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per ilogram of plasma calculated (Table 13).

Table 13 Characterisation summary of solubilised Fraction 1 paste Sample Protein Fibrinogen Total fibrinogen 96 clottable Fibrinogen mg/niL (mg protein gkplasma SFP 15.90 10.34 509 65 0.71 -GASH' 9.73 8.21 191 84 0.27 GASH' 250 mM'v 9.60 7.14 171 74 0.24 8-ACA WO 01/48016 PCT/AU00/01585 Characterisation of in process samples generated after extraction of frozen fraction 1 paste in buffer containing 1 IU/mL ATIII showed very similar results with respect to clottable protein and yield to those obtained from previous extractions ATm.

Stability at 37C SDS-PAGE analysis of in process stability samples from each extraction experiment was performed.

The stability (in hrs) of solubilised fraction 1 paste, Al(OH), absorbed and Gly/NaCl precipitated samples from fresh and frozen (-80 0 C) paste, extracted in buffers with and without ATII are detailed in Table 14 below.

Table 14 Stability of fibrinogen (hours) 4 0 C -80 0

C

-ATIII +ATII -ATIII +ATIII SFP 40 74 >112 >112 ASFP 63.5 92 >112 >112 GASFP >92 >112 >112 >112 GASFP >112 >112 >112 >112 250 mM eACA Analysis of frozenlthawed/resolubilised GASFP The frozen Gly/NaC1 precipitate was thawed at 37 0 C and resolubilised using Buffer D 100 mM eACA at 30 0 C. The precipitate resolubilised within min. Samples were then assayed for total and clottable protein in Table below and for stability at 37 0

C.

WOO01/48016 PTAO/18 PCT/AUOO/01585 31 Table Thawed and resolubilised Gly/NaCl precipitate GASFP Protein Fibrinogen Total fibrinogen clottable Fibrinogen (mg/inL) (mg) protein g~gpa Extraction 1 16.21 14.18 277 87 0.39 Extraction 2 12.80 11.18 206 87 0.29 ATMI) Extraction 3 12.74 10.91 195 86 0.27 Extraction 4 8.81 7.65 134 87 0.19 ATMI) Protein characterisation shows that the freezing of the precipitate does not affect the levels of clottable protein or yield of fibrinogenlkg plasma in the resolubilised Gly/NaCI precipitate.

Stability analysis of the thawed resolubilised Gly/NaGL precipitates showed that the fibrinogen from all extraction conditions was stable for 120 hours. This result is consistent with that of the non-frozen Gly/NaCI precipitates stability.

1.2.6 Addition of heparin to extraction buffer Fresh fraction 1 paste (6g) was extracted in 50 g of the appropriate extraction buffer, treated with AI(OH), and precipitated using Gly/NaCI buffer. The precipitates were then split in half. Half of the precipitate was stored at -80 0 C. The other half was resolubilised using Buffer D. The resolubilised precipitates were then split in half again and 250 mM sAGA added to one half and no eAGA added to the other half. The samples were then assessed for stability at 371C.

Protein Characterisation Extdraction 1 Fresh fraction 1 paste (6.0g) was extracted in 50.0 g of extraction buffer containing 20 mM NaCitrate, 5 mM sAGA, 0.4 M Na~i, 20 IU/rnL heparin, pH WO 01/48016 PCT/AUOO00/01585 32 7.3. The solubilised fraction 1 paste was then treated with AI(OH) 3 and precipitated using Gly/NaC1 buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma was calculated (Table 16).

Table 16 Characterisation summary Sample Protein Fibrinogen Total fibrinogen clottable Fibrinogen m/mL mg/mL (mg) protein g/g plasma SFP 27 18.09 974 67 1.67 ASFP 20.19 12.96 718 64 1.23 GASFP 27.29 24.11 493 88 0.85 GASFP+ 250 mM 27.79 24.52 517 88 0.89 sACA_ Characterisation of in process samples generated from extraction with buffer containing 20 IU/mL heparin were very similar to those obtained with the original extraction buffer. Clottable protein was again 67% after extraction and increased to 88% after Gly/NaCI precipitation. The yield was high with 1.67 g fibrinogen extracted per kilogram of plasma.

Extraction 2 Fresh fraction 1 paste (6.02g) was extracted in 50.17 g buffer containing 20 mM NaCitrate, 5 mM ACA, 0.4 M NaCl, 60 IU/mL heparin, pH 7.3. The solubilised fraction 1 paste was treated with Al(OH), and then precipitated using Gly/NaCl buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma was calculated (Table 17).

1. WVO01/48016 PTAO/18 PCT/AUOO/01585 33 Table 17 Characterisation summary Sample Protein Fibrinogen Total fibrinogen clottable Fibrinogen (mroeig)k pa SFP 24.12 16.09 858 67 1.47 ASFP 19.56 13.04 707 67 1.21 GASFP 25.89 23.00 440 89 0.75 GASFP+ 250 mM 25.64 22.82 451 89 0.77 &AGA Characterisation of in process samples from fraction 1 paste extracted with buffer containing 60 JIJ/mL heparin showed results very similar to the original extraction buffer in terms of total and clottable protein and fibrinogen yield.

Extraction 3 Fresh fraction 1 paste (6.03g) was extracted in 50.25 g buffer containing 20 mM NaCitrate, 5 m.M &ACA, 0.4 M NaCi, 20 IU/mL heparin, 1 rU/mL ATm, pH 7.3. The solubilised fraction 1 paste was then treated with Al(OH), and precipitated using Gly/NaCi buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma was calculated (Table 18).

WO 01/48016 WO 0148016PCT/AUOO/01585 34 Table 18 Characterisation summary Sample Protein Fibrinogen Total fibrinogen 0, clottable Fibrinogen ng/niL mg/niL protein glgpa SFP 17.92 11.43 592 64 1.01 ASH' 12.97 .7.97 419 61 0.72 GASFP 27.16 24.41 439 90 0.75 GASFP 250 mM 26.27 23.25 431 88 0.73 cAGA__ Characterisation of in process samples from fraction 1 paste extracted in buffer containing 20 HJ/mL heparin and ATM showed results very similar to the those 'obtained with the original extraction buffer. The yield was lower with 1 g fibrinogen extracted per ilogramn of plasma.

Extraction 4 Fresh fraction 1 paste (6.01g) was extracted in 50.09 g buffer containing 20 mM NaCitrate, 5 mM eACA, 0.4 M Na~l, 60 IU/mL heparin, 1 IUimL ATM, pH 7.3. The solubilised fraction 1 paste was then treated with Al(OH) 3 and then precipitated using Cly/NaCl buffer.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per ilogram of plasma was calculated (Table 19).

WO 01/48016 PTAO/18 PCT/AUOO/01585 Table 19 Characterisation summary Sample Protein Fibrinogen Total fibrinogen 96 clottable Fibrinogen na nL (mg protein g1Kg plasma SEP 23.20 14.60 774 63 1.33 ASFP 18.42 12.13 659 66 1.13 GASFP 18.93 16.79 281 89 0.48 GASFP 250 mM 19.94 17.60 305 88 0.52 FAGA Once again the results of in process characterisation of total and clottable protein from solubilise fraction 1 paste to Gly/Na~i precipitate were consistent. A significant loss of yield over the Gly/h.TaCl precipitation stage was noted. Based on previous result, however, the presence of heparin and ATLII is not thought to contribute to the loss over this step.

Stability at 37-C The stability of solubilised fraction 1 paste, Al(OH) 3 absorbed and Gly/NaCi precipitated samples from fresh paste, extracted in buffers containing 20 IU/rnL or 60 ITJ/mL heparin, with and without ATI are detailed in Table 20 below.

WOO01/48016 PTAO/18 PCT/AUOO/01585 36 Table Stability of fibrinogen IIJ/mL -ATDI +ATMl -ATMI +ATMI W4, extraction buffer SFP 23 hrs 120 hrs 23 hrs 72 hrs at 39 _______clotted at 63 ASFP 39 72 46 72 GASFP 23 63 120 120 clotted at 39 clotted at 72 GASFP 120 120 120 120 1250 mM FaACA The presence of ATMT in the extraction buffer appears to increase the stability of the samples at 37*C. The presence of 60 IU/rnL heparin appears to further enhance this stability to the level obtained after the addition of 250 mM &ACA.

Analysis of frozenithawed/resolubilised GASFP The frozen Gly/NaCI precipitate was thawed at 37 0 C and resolubilised using Buffer D 100 mnM eACA at 30 0 C. The precipitate resolubilised within min. Samples were then assayed for total and clottable protein in Table 21 below and for stability at 37 0

C.

Table 21 Thawed resolubilised Gly/NJaCI precipitate GASFP Protein FIbrinogen Total fibrinogen Glottable Fibrinogen .(Mgj (Mg) protein ()gkplasma Extraction 1 29.08 25.86 548 89 0.94 Extraction 2 28.43 25.60 555 90 0.95 Extraction 3 28.05 25.35 542 90 0.92 Extraction 4 18.00 16.00 330 89 0.56 WO 01/48016 PCT/AU00/01585 37 Protein characterisation shows that the freezing of the precipitate does not affect the levels of clottable protein or yield in the resolubilised Gly/NaCl precipitate. The yield from Gly/NaCI precipitate of Extraction 4 was low but this correlated to the low yield seen in the non-frozen GASFP.

Stability analysis of the thawed resolubilised Gly/NaCl precipitates showed that the fibrinogen from Extraction 1 was stable for at least 36 hrs (last time point), Extraction 2 was stable for at least 72 hrs (last time point), Extraction 3 was stable for at least 72 hrs (last time point), and Extraction 4 was stable for 96 hrs. This result is consistent with that of the non-frozen Gly/NaCl precipitates stability.

1.2.7 Concentration study Concentration study 1 1.5 g, 3 g, 4.5 g, 6 g, 7.5 g 9 g of fresh fraction 1 paste were resolubilised in 50 g of extraction buffer containing 0.8 M NaC1.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table 22).

WO 01/48016 WO 0148016PCTIAUOO/01585 38 Table 22 Characterisation summary of solubilised Fraction 1 paste Sample Mass of Protein Fibrinogen Total 0,clottable Fibrinogen paste mglmL mg/mL fibrinogen (mg) protein gfKg plasma ratio SFF #1 1.5 6.57 4.72 233 72 1.38 (1:33.3) SFP #2 3 12.57 8.51 433 68 1.29 (1:26.7) SF? #3 4.49 32.50 23.40 1287 72 2.56 11. 1) SFP #4 6.03 25.49 16.76 892 66 1.32 SFP #5 7.51 30.84 21.83 1202 71 1.43 #6 9.01 19.59 13.3 693 68 0.69 Regardless of the fraction 1 paste to buffer ratio the levels of clottable protein extracted were similar (see Figure 1).

The yield of fibrinogen extracted per kilogram of plasma, however, is not consistent (see Figure 2).

The maximum yield in this experiment was obtained when a paste to buffer ratio of 1:11.1 was used. After the 2 hoar extraction period it was noted that the extraction of 1.5 g, 3 g and 4.5 g fraction 1 paste were completely resolubilised but the extractions of 6 g, 7.5 g and 9 g fraction 1 paste were not. This may suggest that when 4.5 g fraction 1 paste in 50 mL buffer (1:11.1) is extracted completely the maximum levels of fibrinogen are extracted. When the ratio of paste to extraction buffer ratio is increased, not all the fibrinogen present is extracted. Alternatively, the difference in yield of fibrinogen per kilogram of plasma may be due to the non-homogeneous nature of the starting material.

WO 01/48016 WO 0148016PCT/AUOO/01585 39 Concentration Study 2 g, 9 g, 13.5 g, 18 g, 22.5 g 27 g were resolubilised in 150 mL of extraction buffer containing 0.8 M Na~i, 60 IU/mL heparin at 37 0 C for minutes.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table 23).

Table 23 Characterisation summary of solubilised fraction 1 paste #2 Sample Mass of Protein Fibrinogen Total fibrinogen %A clottable Fibrinogen paste g mg/mi mg/mL (ing) protein glkg plasma ratio SFP #1 4.51 7.08 4.71 706 66 1.21 (1:33.3) SFP #2 9.01 13.45 8.81 1362 65 1.17 (1:26.7) SF? #3 13.50 20.00 13.10 2073 66 1.19 1(1: 11. 1) SF? #4 18.02 25.12 16.14 2633 64 1.13 SF? #5 22.51 31.99 20.31 3401 63 1.17 SF? #6 27.01 35.55 22.04 3787 62 1.08 1 In this experiment it was noted that after the 90 min. extraction period all fraction 1 paste samples had been completely solubilised.

Regardless of the fraction 1 paste to buffer ratio, the levels of clottable protein extracted were similar (see Table 23).

The yield of fibrinogen extracted per kilogram of plasma was also unchanged over the range of paste to buffer ratios (Figure This suggests that the inconsistent results of the previous experiment (Figure 2) could be attributed to the non-homogeneous nature of the heparin paste. Furthermore, WO 01/48016 PCT/AU00/01585 this data indicates that a higher paste to buffer ratio could be used in the initial extraction resulting in smaller solubilised fraction 1 paste volumes.

1.2.8 Extraction temperature study Fresh fraction 1 paste (18g) was extracted in 150 mL of buffer (20 mM NaCitrate, 5 mM EACA, 0.8 M NaCl, pH 7.3) at a ratio of 1g:8.33g buffer for 2 hours at room temperature and at 37°C. Samples were taken at 30 min., min., 90 min. and 120 min. throughout extraction. The solubilised paste was then centrifuged and another sample taken (125 min).

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table 24).

Table 24 Characterisation summary of solubilised Fraction 1 paste Sample Mass of Protein Fibrinogen Total Fibrinogen paste mg/mL mg/mL fibrinogen (mg) clottable g/Kg protein plasma SFP RT 30' 17.99 8.97 6.58 1011 73 0.50 SFP RT 60' 13.06 9350 1460 72 0.72 SFP RT 90' 17.87 12.88 1980 72 0.98 SFP RT 120' 19.62 13.80 2121 70 1.05 SFP RT 125' 20.98 15.18 2333 72 1.16 SFP 37 0 C 30' 18.02 22.52 15.61 2568 69 1.27 SFP 37 0 C 60' 24.66 16.79 2762 68 1.37 SFP 37 0 C 90' 27.87 19.19 3157 69 1.56 SFP 37 0 C 120' 27.15 18.42 3030 68 1.50 SFP 37 0 C 125' 26.32 17.75 2920 67 1.44 Total and clottable protein extracted at RT and at 37 0 C over time was unchanged (see Figure 4).

However, the yield calculations showed that the level of fibrinogen extracted from fraction 1 paste extracted at 37°C was higher than that extracted at room temperature at all time points. It was also noted that fraction 1 paste was not completely resolubilised after 2 hr extraction at room WO 01/48016 PCT/AUOO/01585 41 temperature but it was completely resolubilised after 2 hr extraction at 37 0

C

(see Figure 5 below).

1.2.9 Production scale extraction Extraction 1 Fresh fraction 1 paste (20.0 kg) was extracted at a ratio of 1 g paste 8.33 g buffer in 20 mM NaCitrate, 5 mM sACA, 0.8 M NaC1, 60 IU/mL heparin, pH 7.3, at 37°C for 90 min.

Solubilised fraction 1 paste was treated with Al(OH), and Gly/NaC1 precipitation was performed. The precipitate was split into two and half the precipitate resolubilised in Buffer D containing 100 mM sACA and the other half frozen at -80°C. Half of this resolubilised Gly/NaCl precipitate was then treated with SD, applied to the MacroPrep column. The eluate was collected, sampled and frozen at -80 0 C. The other half was treated with SD, wet heat treated and applied to the MacroPrep column. The eluate was collected, sampled and frozen at -80 0 C. The frozen Gly/NaCl pellet was thawed, resolubilised (FTR) and samples for total protein and clottable protein.

All samples were assayed for total protein and clottable protein and the yield of fibrinogen per kilogram of plasma calculated (Table Table Characterisation summary Sample Protein Fibrinogen Total lottable Plasm. ibrinogen mg/mL mg/mL f rinogen (mg) protein (fg/mL) gkg plasma SFP _29.09 18.15 3386790 62 1.78 ASFP 23.11 14.18 32363 1 1_.54 GASFP 25.91 22.66 13376 7 219.6 1.27 GASFP ppt (FTR) 27.74 24.2 14259 7 1.35 Run 1 Eluate 11.94 10.4 770 87 0.66 0.97 Run 2 pre wet heat 1.13 0.92 691 82 0.77 Run 2 post wet heat 1.36 1.11 836 82 0.93 Run 2 wet heat Eluate 2.85 2.58 263 91 0.42 0.30 WO 01/48016 PCT/AU00/01585 42 Characterisation of fraction 1 paste extracted at a production scale showed very similar results to that extracted previously at a small lab. scale.

Approximately 62% clottable protein was extracted at this stage at a yield of 1.78 g fibrinogen per kilogram of plasma. Following Gly/NaCl precipitation the clottable protein again increased to greater than 85%, as expected.

(Analysis of the frozen Gly/NaCl precipitate after thawing and resolubilisation showed no loss of total protein, clottable protein or yield of fibrinogen per kg plasma). The material eluted off the column was also high in clottable protein and yield. The plasminogen level of this eluate was very low at 0.66 4tg/mL which equates to a 160 fold reduction in the level of plasminogen across this step.

Resolubilised Gly/NaC1 precipitate that was wet heat treated and applied to the ion exchange column was high in clottable protein before and after the wet heat treatment. No loss of fibrinogen was seen over the wet heat treatment step. Wet heat treated material, after elution from the ion exchange column, was high in clottable protein but only 30%/ of the fibrinogen was recovered. Plasminogen levels were also low at 0.42 pg/mL.

The results of the ion exchange chromatography of GASFP and wet heat treated GASFP show that the column is acting efficiently to remove plasminogen from the product.

SDS-PAGE analysis All samples rich in fibrinogen as seen by the three major bands between 45 and 66 kDa under reducing conditions.

Stability at 37°C Stability of in process samples was similar to previous findings (See Appendix for Stability gels). The solubilised fraction 1 paste was stable for approximately 15 hrs. This sample clotted before 39 hrs at 37°C. After Al(OH) 3 absorption, the fibrinogen molecule was stable for 48 hrs and after Gly/NaCl precipitation, for >70 hrs. Following elution from the ion exchange column the wet heat treated and non-wet heat treated fibrinogen was stable for at least 113 hrs.

WO 01/48016 PCT/AU00/01585 43 Extraction 2 Fresh fraction 1 paste (30.0 kg) was extracted at a ratio of 1 g paste 8.33 g buffer in 20 mM NaCitrate, 5 mM eACA, 0.8 M NaCI, 60 IU/mL heparin, pH 7.3, at 37 0 c for 90 min.

Total protein and clottable protein were determined and yield per kilogram of plasma calculated in Table 26 below.

Table 26 Characterisation of solubilised fraction 1 paste Total F1 paste Fl paste Extracted Fibrinogen Total Mass of Fibrinogen generated extracted F1 paste fibrinogen starting plasma YIELD (mg/mL) (kg) (glkg SI plasma) 59730 30000 280000 20.64 5779.2 7728 1.49 The fraction 1 paste extracted in this experiment was from the same batch as used in the above section entitled "Concentration Study At a small scale, the average yield of fibrinogen per kilogram of plasma was 1.16.

At a scale greater than 1,000 times this lab scale the yield is shown to be 1.49 g/kg plasma. This further suggests that the lab scale is not representative of the expected yield at production scale as a result of the non-homogeneous nature of the fraction 1 paste.

The results of the two production scale extractions show that the expected yield of fibrinogen per kilogram of plasma is 1.5 1.8 g/kg.

1.2.10 Comparison of fraction 1 paste and heparin paste as the starting materialforfibrinogen purification Data gained from experiments detailed in this report is summarised in Table 27 below.

WO 01/48016 PCT/AU00/01585 44 Table 27 Comparison of yield from fresh fraction 1 paste and heparin paste extracted at a ratio of 6 g paste to 50 mL buffer Yield Stability Sample Fraction 1 Heparin paste Fraction 1 Heparin paste Extracted 1.5 0.42 24 <24 A1(OH)3 1.32 N/A 48 N/A Gly/NaCl 0.96 0.34 >70 72 Post Wet Heat 0.85 0.26 54 Post Wet Heat column 0.64* 0.23 >113 120 eluate Concentrate 0.58* 0.21 >113** 120 based on expected losses Comparison of fraction 1 paste to heparin paste shows a significant increase in yield of fibrinogen generated per kg plasma.

Comparison of fraction 1 paste to heparin paste in terms of stability shows that stability of fibrinogen increases throughout both processes. Final fibrinogen concentrate is equally stable regardless of the starting material.

1.3 Discussion Fibrinogen was initially extracted from fraction 1 paste at a small scale. Experiments were performed to assess the effect of adding ATIII and heparin to the buffer, and to assess the effect of temperature on the extraction procedure. Results showed that the presence of heparin in the extraction buffer increased the stability of the fibrinogen molecule at this and subsequent steps of the process. Equal stability can also be attained by the addition of at least 125 mM eACA at the resolubilised Gly/NaCl stage.

Performing the extraction at 37 0 C was shown to increase the rate of extraction of fibrinogen and the yield of fibrinogen per kilogram of plasma.

Thus, the extraction conditions recommended for fraction 1 paste are 20 mM Tri-sodium citrate, 0.8 M NaC1, 5 mM sACA, 60 IU/mL heparin, pH 7.3, extracted for 90 minutes at 37 0

C.

WO 01/48016 PCT/AU00/01585 At a production scale, the extraction of fraction 1 paste was performed on a scale greater than 750 times that of the lab scale. In these large scale experiments, the fraction 1 paste was extracted with the optimised buffer (containing heparin and performed at 37°C) and the resultant yields were 1.78 g/kg plasma and 1.49 g/kg plasma. The same batch of fraction 1 paste was extracted at both lab and production scale under identical extraction conditions and the yields obtained were 1.15 g/kg and 1.49 g/kg, respectively.

With an expected yield of 1.5 g fibrinogen per kilogram of plasma at the solubilised fraction 1 paste stage, the yield from fraction 1 paste is significantly higher that that extracted from heparin paste (0.42 g/kg plasma).

Variation of the fraction 1 paste to extraction buffer ratio suggested in the first small scale experiment that 4.5 g:50 mL buffer (a ratio of 1:11.1) was optimal to obtain the highest yield/kg plasma. However, in a subsequent experiment performed at three times this scale and with the improved extraction buffer, even at the highest paste to buffer ratio all the fibrinogen was extracted. This result suggests that greater masses of fraction 1 paste may be solubilised in extraction buffer compared to heparin paste (1:8.33) which will result in smaller total extraction volumes.

The protein characterisation of solubilised fraction 1 paste showed similarities and differences to solubilised heparin paste. The amount of clottable protein obtained from either starting material is similar at approximately 65%. Levels of plasminogen and factor XIII were higher in solubilised fraction 1 paste than those extracted from heparin paste, however, the level of fibronectin was significantly lower. When the solubilised fraction 1 paste was further processed using the heparin paste method the material behaved in a similar manner to heparin paste material over the subsequent purification steps. Alhydrogel absorption demonstrated the reduction of factor II to undetectable levels that correlated with an increase in fibrinogen stability at 37C. Gly/NaC1 precipitation resulted in the purification of fibrinogen to greater than 80% clottable protein and the reduction of fibronectin to negligible levels. Ion exchange chromatography was shown to reduce plasminogen to negligible levels in the eluate and increase the stability of the fibrinogen to approximately 120 hrs which is equivalent to heparin paste eluate stability.

As fraction 1 paste is a by-product of another production process it is advantageous to hold the product at this stage prior to commencing the WO 01/48016 PCT/AU00/01585 46 fibrinogen manufacturing process. Heparin paste, a by-product of factor VIII concentrate can be stored frozen for up to 13 months at -80°C without affecting the resultant levels of clottable protein once extracted.

The stability of frozen fraction 1 paste as a starting material is promising. After processing as far as the ion exchange column, the product demonstrated excellent stability (>208 hrs). In a subsequent experiment (Section frozen fraction 1 paste was extracted in the presence and absence of ATmI. No handling problems were encountered after extraction of frozen fraction 1 paste, in either buffer, and the stability of SFP and ASFP was far greater than that observed for previous or subsequent experiments.

Experiments were also performed to assess the possibility of holding the process at the Gly/NaCl precipitate stage prior to resolubilisation. Results of Gly/NaCl pellet, frozen at -80°C and subsequently thawed and resolubilised, showed that this hold point did not compromise product quality with respect to clottable protein, stability or yield.

The results presented herein show that Fraction I paste is a suitable starting material for the purification of fibrinogen, and has the potential to increase the yield of final product three fold compared to heparin paste.

EXAMPLE 2: Separation of fibrinogen from plasminogen using ion- Sexchange chromatography 2.1 Materials and Methods 2.1.1 Sample Preparation A Gly/NaC1 precepitate was obtained from cryoprecipitate using a modification of the methods described in Winkleman et al., 1989. Initially, the frozen resolubilised Gly/NaCl precipitate (-80 was thawed in a waterbath at 30 OC. To 40g of resolubilised Gly/NaCl precipitate was added 2.19g of stock detergent solution and 132 mg of TNBP. The sample was then diluted using sample dilution buffer (25 mM Tris, pH 8.0) until the conductivity was below 10.5 mS/cm. Finally, the sample was filtered through a 0.8 ujm membrane filter. Each sample was prepared immediately prior to the start of each run. Failure to dilute the sample often results in a large unbound peak i.e. some fibrinogen is eluted in the unbound.

WO 01/4-8016 WO 0148016PCT/A UOO/O 1585 47 2.1.2 MacroPrep HQ Purification The following chromatographic conditions were used to purify the resolubilised gly/NaCi paste: Bed Height approx. 20 cm Column Volume approx. 100 ml Flow rate 10 mI/min 113 cm/br) Detection LTV 280 nm Chromatographic Method Equilibration: 1.5 CV of MQ buffer and when conductivity (post-column) is 90-110% of the prepared buffer.

Load Sample Wash: 6 CV of MQ buffer.

Elution. ME buffer Buffer D 200 mM NaCi, pH7.O Regeneration: 2 CV of 1 M Na~i 2.2 Results 2.2.1 Purification Using Wash Buffer (MQ 25mM Tris, 100 nM NaC1, pH Duplicate runs were performed using 25mM Tris, 100 mM NaCi, pH as the wash (MQ) buffer. Samples were loaded on a MacroPrep column and the collected fractions analysed. Results are shown in Table 28.

Table 28 MQ 25mM Tris 100 mM MaCI, pH padl TotRI ProU, ft~bla Ptvtda Phda NWICIObhc tb o1Uofr Twa.I w d0 %reo %Ckft1bloP,'gdn PWSMI. 4.62 326 0.952 0.54 405 90 Sr/% 68% 94Y. 184% Tubelp 1.6 0D86 0.767 0.51 1121 10 7.4% 54% 76IM ?fl9094 2.3 0UD75 0.408 0.52 198 50 64% 9.0% 49% Pad 95,100 1.44 0.7 "0.2 0.49 7000 00 71% 9.5% 4W% 67-4 pas! 111-106 1.25 0.62 40.2 0.45 T 20 60 77% 9s% 5W% h. il8 9.75 8.366 .5 59 0 63% 39% 90% 90% Po-W 2,64 2.811 A 05 30 70% 179% 71% 86% PrW "M 1.67 0. D92 OX 1 0.46 1957 00 7% 55% 82% Iod%.IOS 1.38 0 .65 <8.2 1 0.47 V5 1% 2 0.2% 47% 79% The average recovery of fibrinogen was 82% and plasmiinogen was 10%. These figures were used as a bench mark to judge the success of any further modifications to the process.

WO 01/48016 PCT/AU00/01585 48 2.2.2 Addition of EACA to Load Sample.

With large scale production, not all of the samples can be processed in a single ion exchange run and hence some diluted samples were left at room temperature while other samples are purified. It was discovered that the samples were breaking down during this period.

The addition of 100 mM e-amino caproic acid, EACA (in respect to the volume of undiluted resolubilised Gly/NaCl paste) to the sample increased the stability of the sample from between 0-15 hr to 15-23 hr. There were no significant changes in the chromatographic profile and the recovery of fibrinogen was 93%.

WO 01/48016 PCTAU00/01585 49 2.2.3 Addition of EACA and Lysine to Wash (MQJ Buffer.

The following wash buffers were used in the purification protocol: (i) mM Tris, 20 mM Lysine, 100 mM NaCI, pH 8.0 and (ii) 50 mM Tris, mM EACA, 100 mM NaCI, pH 8.0. Samples were purified using the Macroprep column and the collected fractions compared. Results are shown in Table 29 below.

Table 29 Addition of EACA Lys to MQ Buffer

LO

Bller Prote'in No Clonttable Cottable Protosn %ProPmtehn Clottale Stabliy rgImI mmI ml as Cottable Reovey* (hs) #14 l0b Fration 1 20mM Lys. 50 mM Tris 10.08 0.76 9.30 92% 48-71 Frec tio n 2 100 mM NaCi pH 8.0 2.30 0.37 1.94 84% 51.67% 48-71 Fra c tion 3 1.84 0.32 1.32 80% N/A Fraction 4 2.00 0.14 1.92 93% N/A Fr c tio n I 20 mMLys, 50 mM Tris 9.58 0.73 6.5 92% 48-71 Frtion 2 100 mM NaCI, pH 8.0 1.56 0.29 1.37 83% 53.19% 48-71 ro io n 3 1.71 0.18 1.52 89% N/A 16 Fra c Dt 1R 20 mM Lys, 50 mM Tlris 9.50 0.77 8.73 92% 48-71 Fra c tio 2 100 mM NaCI, 8.0 2.88 0.40 2.46 86% 49.11% 41-78 Fra ti n 3 1.83 0.32 1.31 80% N/A frclon 4 1.89 0.18 1.71 90% N/A I Os Fr c tia 20 mM EACA 5 mM Tris 13.27 0.80 12.47 94% 71-1 Fractio a 2 100 mM Na, PH 8.0 2.65 0.35 2.30 87% 72.12% 71-91 Fraction 3 1.57 0.20 1.28 81% N/A Fraction 4 1.51 0.08 1.45 96% N/A l0b Fraction 1 20 mM EACA, S0 mM Tris 13.02 0.77 12.24 94% 71-91 Fractio a 2 100 mM NCI, pH 8.0 2.51 0.38 2.15 86% 70.41% 71-91 Froctitn 3 1.81 029 1.32 82% N/A Fraction 4 1.52 0.05 1.47 97% N/A NB: In these trials EACA was not added to the sample.

With the addition of 20 mM EACA and 25 mM Tris to the MQ buffer, the stability of the collected fractions increased to between 71-91 hours and the recovery of clottable protein was 71.3%. The longer stability could possibly be attributed to the removal of plasminogen. The unbound region of the chromatogram showed a small UV absorbance.

WO 01/48016 PCT/AUOO/01585 The average clottable protein recovery decreased to 51.3% when mM lysine and 25 mM Tris were added to the MQ buffer. In the chromatographic profile, the absorbance during the wash step was larger than that obtained using MQ. Hence it can be assumed that the addition of lysine and tris to MQ caused fibrinogen to elute in the unbound peak. The stability of the collected fractions were between 48-71 hours.

These results show that the addition of EACA and an increase of tris concentration in MQ increased the recovery of clottable protein and stability of column eluate.

2.2.4: Varying MQ Buffer Composition The aim of this experiment was to investigate the effectiveness of the addition of lysine and EACA to the MQ buffer and to examine the effect of the addition of EACA to the sample. Table 30 summarises the Fraction 1 results.

Table Results of Varying Wash Buffer Composition Sample EACA in Buffer Protein recovery Plasminogen Stability Sample MQ plus ug/mi Hrs HPPC04 No 77 5 24 HPPCO6 No 78 23 24-47 HPPC06 No Lys Ts 48 0.25 47 HPPCO4 Yes Lys &Tris 47 <0.5 41 HPPCO6 Yes Lys Tris 49 0.27 68-93 HPPC04 Yes EACA Tris 68 <1.0 113-143 HPPCO6 Yes EACA Tris 79 0.23 >141 HPPC06 Yes 25 mM Tris 77 0.8 66 These results show that when no EACA was added to the sample, and MQ (25mM Tris, 100 mM NaC1, pH 8.0) was used as the wash buffer, the average protein recovery of fraction 1 for HPPC04 HPPC06 was 77% 78% respectively. The stability was approximately 24 hours for HPPC04 and <47 hours for HPPCO6.

WO 01/48016 PCT/AU00/01585 51 Where 25 mM Tris was added to the MQ buffer, the protein recovery was similar to that obtained using MQ but the stability of the collected fraction was increased to 66 hours.

With the addition of lysine to the MQ buffer (50mM Tris, 20 mM Lysine 100 mM NaCI, pH the average protein recovery of fraction 1 for HPPC06 was 48% and the stability was 47 hours. The plasminogen recovery was reduced dramatically from 10.2% to less than 0.5 When EACA was added to the sample prior to loading onto the Macroprep column, and the wash buffer was MQ with lysine,the protein and plasminogen recoveries were approximately the same as those obtained where EACA was not added to the sample. The stability of the eluate, however, was increased from <47 hours to between 68-93 hours for HPPC06 and 41 hours for HPPC04.

Where EACA was added to the samples and the wash buffer was Tris, 20 mM EACA 100 mM NaCL, pH 8.0, the protein recoveries were similar to those obtained when using MQ i.e 68% for HPPC04 and 79% for HPPC06.

The significant difference was the stability of the collected fractions. The stability was in excess of 113 hours as compared to approximately 24 hours when using MQ buffer.

These results show that the wash buffer containing 50mM Tris, 20 mM EACA 100 mM NaCI, pH 8.0 gave good results. In particular, the collected fraction had high protein recovery, low plasminogen recovery and long stability.

2.2.5 Stability of MacroPrep Fractions, Individually and Pooled.

Previously, four fractions were collected from the MacroPrep eluate.

The following experiment was designed to determine whether the fractions can be pooled in order to increase recovery. The collected fractions were placed on stability both individually and pooled in the ratio as if one fraction was collected.

The purification method involved the use of 50mM Tris, 20 mM EACA 100 mM NaCl as the wash buffer and EACA was added to the samples prior to loading on the MacroPrep column. The results are shown in Table 31.

In general, Table 31 shows that the first fraction is generally more stable than the later fractions. This is most probably due to the later fractions being considerably less concentrated than fraction 1 and not Table 31 Results of Fraction Pooling I Prortn Sample I Praction ITota Protein flotnbocProtein Plasininogen Total aecovry j 3jml rngtl I ult I M I Clottable Dotein .rfiroee.

r Rco 36.9 1 1.5% I 13578 stability Hrs 46-7 <22 <22 I'v1e IR, 107 I 20.79 1 3% 1 20.4d 3% 1 98% 0.62 1 0.1 3.3 1 0.1 I 6200 I Unbound1 1.61 _1 0.75 0.48 I 54.74] 7%4 25.5 4% 1 47% 0.1 1 50.2 1 9% I 21.08 42% 93% 0.53 14.71 1 13.67 16.25 0.99 1382 1 46-71 P1.243 3.73 1 3.35 1 782.4 I 1 704.2 I I 2494.5

S

3 1.07 1 0.77 0.1 1 115.56 1 15% I 83.16 1 12% 721 1 10.8 1 0.4% 1 7700 46-71 22-46 <71 <71 71-95 71 19:47 1 I 1 09% 1.19 1.28 1953.3 15853 P19 114 I 53.7 1 M -1 i 9.02 8.45 0.1 9 595.32 1 79% 1 57.7L 93V 94% 1 0.5.4 1 8000 F <23 0.6 0.58 0.6 1.44 0.1 1 46.08 I 81A 1 19.21 1 42%' I 3.2 1 0.2% 6000 NIA I 21.90 7277 581.56 6.32 578.7 1 94% 6.7 1 0.4% j 81600 1 >119 2 1 1.76 1 1.76 -1 0.1 0.2% 1 17600 23-47 3 1 1 1 0.1 97 1 13% I 97 17% 1 100% I 9.7 1 0.5% 10000 <23 I 0.6 I 0.1 19.2 -L 3% 19.2 3% 100% 3.2 6000 .2321 9142 6.37 6.03 0.10 j1 9 .12 13.08 1 93v. 0-30 60267 I .243 3.69 3.51 0.10 22.12 21.08 95,14 0.60 35133 P1 344 3.25 3.10 0.10 22.72 21.68 95% 0.10 30971 95.119 00 Samples F1711a &Fl7_11b used resolubilised Gly/NaC precipitate Batch No. HPPCO4 and F 9_11 a Fl 91 lb used resolubilised Gly/NaC precipitate Batch No. HPPCO6.

WO 01/48016 WOOI/8016PCT/AUOO/O1 585 53 because of the composition of the fractions. This was further examined by pooling the fractions at the same ratio as they were eluted from the column.

When the four fractions were pooled the stability was equivalent to that of fraction 1. By pooling all four fractions, the recovery of protein was increased by approximately 2.2.6 Modification of the El uti on Buffer Results described in 2.2.5 above show that the fractions eluting from the MacroPrep column can be pooled. The profile of the bound fraction shows a large peak with tailing and then a second peak when the column is washed with 1JM Na~i. The second peak is the compression of the tailing due to the higher ionic strength of the -1M NaCl. It was decided to increase the ionic strength of the elution buffer to elute fibrinogen as a single peak.

The concentration of NaCI in the ME buffer was therefore increased from 300 mM to 500 mM, 750 mM and 1M. Results are shown in Table 32.

Table 32 Increasing NaCI Concentration in ME Buffer Totd Miudn (2 tt~ protin Sanis NE B~uffer Fracton Total Rcooovay Cmnlaive Total_ CuawvQmulative %Clottable P'ool IStability N&CI Clottable Hr,_ FOS 12& 300mrM 559.5 493.8 1 439.65 79%A 791A 409.26 83% 831/ 93% 93% 2 101.64 19% 97 r/ 89.44 1 PA 101% 87% 92%1 72 F09 12a 500 mM 615 543.2 1 655.6 107% 107% 599.5 110%Y 1 10% 91% 91% P09 1 2b 500um 615.5 432 1 533.9 87% 87% 478.42 83% 88% 90h1 901A >100 FlO 12& 750mM 654.7 577.8 1 498 76% 76% 447.6 7MA 77% M0. 90% >100 2 1 46.9- 7% 83% 34.51 6%Y 83% 74% 88% >100 F31112a IM 61. -54. 1 570.6- 3% 9 516.24 195% -95% 90% 90%A >100 The addition of an extra 200 mM NaCl to the elution buffer, M4E, was sufficient to elute the fibrinogen in a single peak, with very little tailing.

There was no significant difference in the characterization results of the collected fraction. Although, the ME buffer with 750 mM and 1 M NaCi also WO 01/48016 PCT/AU00/01585 54 work, it is preferred that ME with 500 mM be used due to the requirements of the following steps in the purification process.

22.27 Effect of EACA in sample on Column Eluate Stability and Recoveries The aim of this experiment was to compare the bound fractions eluted off the MacroPrep column from samples (resolubilised Gly/NaCI precipitate) containing either 100 or 200 mM EACA.

Results showed that the protein recovery clottable protein plasminogen (both were <0.2 ug/ml) and stability results (both were 7 days) were equivalent for the fractions collected from both samples. In other words, similar results were obtained for resolubilised Gly/NaC1 samples containing 100 or 200 mM eACA.

A preferred fibrinogen purification process incorporating the ionexchange chromatography method described above is shown in Figure 6.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 01/48016 PTAO/18 PCT/AUOO/01585 References Blomback and Blomnback (1956). Ark Kemi. 10:415-43.

Deutsch and Mertz. (1970). Science, 170:1095-6.

Holm at al (1985). Thrombosis Research, 37:165-176.

jakobsen and Kieruif, (1973). Thrombosis Research, 3:145-159.

Kuyas, Haeberli, Walder and Straub, (1990). Thrombosis Haemostasis, 64(3) :439-444.

Mosesson. (1962). Biochim. Biophys. Acta, 57:204-213.

Robbins, Summaria, Elwyn and Barlow. (1965). J. Biol. Chem, 240:541.

Stathakis et al (1978). Thorombosis Research, 13:467-475.

Takeda, (1966). J. Clin. Investigation, 45:103-111.

Vuento et al (1979), Biochemnistry 1, 183:331-337

Claims (39)

1. A method for purifying fibrinogen, the method comprising extracting fibrinogen from a Fraction I precipitate by admixing the Fraction I precipitate with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises salt at a concentration of at least 0.1M and heparin at a concentration of at least 10 IU/ml.

2. A method according to claim 1 wherein the concentration of salt is at least 0.4M.

3. A method according to claim 1 or claim 2 wherein the salt is selected from the group consisting of chloride, phosphate and acetate salts or a combination thereof.

4. A method according to any one of claims 1 to 3 wherein the salt is NaCl. A method according to any one of claims 1 to 4 wherein the concentration of heparin is at least about 20 IU/ml.

6. A method according to any one of claims 1 to 4 wherein the concentration of heparin is at least about 60 IU/ml.

7. A method according to any one of claims 1 to 6 wherein the extraction buffer further comprises Tri-sodium citrate at a concentration of about

8. A method according to any one of claims 1 to 7 wherein the extraction buffer further comprises at least one co-amino acid.

9. A method according to claim 8 wherein the at least one co-amino acid is present in the extraction buffer at a concentration of at least WO 01/48016 PCT/AUOO/01585 57 A method according to any one of claims 1 to 9 wherein the extraction buffer further comprises antithrombin n (ATIII) at a concentration of at least about 1 IU/ml.

11. A method according to any one of claims 1 to 10 wherein the extraction buffer comprises Tri-sodium citrate at a concentration of about NaCl at a concentration of about 0.8M, heparin at a concentration of about 60 IU/ml and at least one co-amino acid at a concentration of about

12. A method according to any one of claims 1 to 11 wherein the extraction buffer has a pH of about 7.3.

13. A method according to any one of claims 1 to 12 wherein the extraction of fibrinogen is performed at about 37 0 C.

14. A method according to any one of claims 1 to 13, the method further comprising the step of incubating the extracted fibrinogen in solution with aluminium hydroxide followed by centrifugation and removal of the precipitate. A method according to any one of claims 1 to 14, the method further comprising the step of precipitating the fibrinogen in the extracted fibrinogen solution by the addition of glycine and NaC1.

16. A method according to claim 15, the method further comprising the step of resolubilising the fibrinogen precipitate in a buffer comprising NaCl at a concentration of around 100mM, CaCl 2 at a concentration of around 1.1M, Na-citrate at a concentration of around 10mM, tris at a concentration of around 10mM and sucrose at a concentration of around 45mM, with a pH of about 6.9.

17. A method according to any one of claims 1 to 16, the method further comprising the steps of: applying the extracted fibrinogen solution to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; WO 01/48016 PCT/AU00/01585 58 eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

18. A method according to claim 17, the method further comprising washing the ion exchange matrix with a buffer comprising at least one a- amino acid prior to eluting the fibrinogen from the matrix.

19. A method of purifying fibrinogen, the method comprising: extracting fibrinogen from a Fraction I precipitate by admixing the Fraction I precipitate with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises salt at a concentration of at least 0.1M; precipitating the fibrinogen; and solubilising the fibrinogen in a solution comprising at least one o-amino acid at a concentration of at least 100mM. A method according to claim 18 wherein the concentration of salt in the extraction buffer is at least 0.4M.

21. A method according to claim 19 or claim 20 wherein the salt is selected from the group consisting of chloride, phosphate and acetate salts, or a combination thereof.

22. A method according to any one of claims 19 to 21 wherein the salt is NaC1.

23. A method according to any one of claims 19 to 22 wherein the buffer further comprises Tri-sodium citrate at a concentration of about

24. A method according to any one of claims 19 to 23 wherein the extraction buffer further comprises heparin at a concentration of at least IU/ml. A method accbrding to any one of claims 19 to 24 wherein the at least one (o-amino acid is present in the extraction buffer at a concentration of at least 59

26. A method according to any one of claims 19 to 25 wherein the extraction buffer comprises Na-citrate at a concentration of about 20 mM, NaCl at a concentration of about 0. 8M and heparin at a concentration of about 60 IU/ml.

27. A method according to any one of claims 19 to 26 wherein the fibrinogen is precipitated in step by the addition of a buffer comprising glycine and NaC1.

28. A method according to any one of claims 19 to 27 wherein the fibrinogen precipitate is solubilised in step using a buffer comprising NaCl at a concentration of around 100mM, CaC12 at a concentration of around 1.1M, Na-citrate at a concentration of around 10mM, tris at a concentration of around 10mM and sucrose at a concentration of around

29. A method according to any one of claims 19 to 28, the method further comprising: 15 applying the fibrinogen solution from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

30. A method according to claim 29, the method further comprising washing the ion exchange matrix with a buffer comprising at least one co-amino acid prior to eluting the fibrinogen from the matrix.

31. A method according to any one of claims 8 to 30 wherein the at least one o- amino acid is e-amino caproic acid (EACA).

32. A method for purifying fibrinogen, which method comprises: extracting fibrinogen from Fraction 1 precipitate by the Fraction 1 precipitate with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises at least one o-amino acid at a concentration of at least WO 01/48016 PCT/AU00/01585 applying the extraction buffer from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate.

33. A method according to claim 32, the method further comprising washing the ion exchange matrix after step with a solution comprising at least one o-amino acid.

34. A method of purifying fibrinogen from a fibrinogen containing solution, the method comprising: applying the solution to an ion exchange matrix, under conditions such that fibrinogen binds to the matrix; washing the ion exchange matrix with a solution comprising at least one o-amino acid; eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate. A method according to any one of claims 32 to 34 wherein the o-amino is e-amino caproic acid (EACA).

36. A method according to any one of claims 32 to 35 wherein the o-amino acid is present in the extraction buffer at a concentration of between 500mM.

37. A method according to claim 36 wherein the co-amino acid is present in the extraction buffer at a concentration of between 50-500mM.

38. A method according to claim 37 wherein the o-amino acid is present in the extraction buffer at a concentration of about 100mM. 61

39. A method according to any one of claims 32 to 38 wherein the fibrinogen containing solution is diluted such that the conductivity is below 10.5 mS/cm before it is applied to the ion exchange matrix.

40. A method according to any one of claims 33 to 39 wherein the buffer used to wash the ion exchange matrix comprises tris at a concentration of about 50mM, (ii) a o-amino acid at a concentration of about 20mM, and NaC1 at a concentration of about

41. A method according to claim 40 wherein the buffer used to wash the ion exchange matrix has a pH of about

42. A method according to claim 40 or claim 41 wherein the buffer used to wash the ion exchange matrix has a conductivity of about 11. ImS/cm.

43. A method according to any one of claims 32 to 42 wherein the fibrinogen is eluted from the matrix in a buffer comprising about 10 mM Tris, 10 mM citrate, mM sucrose; and NaCl at a concentration of between 200mM to 1.OM.

44. A method according to claim 43 wherein the NaCl is at a concentration of about

400-500 mM. A method according to claim 43 or claim 44 wherein the elution buffer has a pH of about 46. A method for purifying fibrinogen, which method comprises: extracting fibrinogen from a fibrinogen containing material by admixing the material with an extraction buffer such that fibrinogen is solubilised in the extraction buffer, wherein the extraction buffer comprises at least one co-amino acid at a concentration of at least applying the extraction buffer from step to an ion exchange matrix under conditions such that fibrinogen binds to the matrix; washing the ion exchange matrix after step with a solution comprising at least one o-amino acid; 35 eluting the fibrinogen from the matrix; and optionally recovering the fibrinogen from the eluate. oo" 62 Dated this twenty-ninth day of September 2004 CSL Limited Patent Attorneys for the Applicant: F B RICE CO off.