WO2019193595A1

WO2019193595A1 - A closed system and methods to produce therapeutic, purified plasma proteins

Info

Publication number: WO2019193595A1
Application number: PCT/IL2019/050387
Authority: WO
Inventors: Yair Pilpel; Mishel NICOLA
Original assignee: Eio Biomedical Ltd.
Priority date: 2018-04-04
Filing date: 2019-04-04
Publication date: 2019-10-10

Abstract

A sterile closed-system for producing a single therapeutic dose of a purified, enriched, therapeutic blood product as well as methods for preparing said therapeutic blood product, are provided.

Description

A CLOSED SYSTEM AND METHODS TO PRODUCE THERAPEUTIC, PURIFIED PLASMA PROTEINS

FIELD OF THE INVENTION

The present invention relates in general to production of therapeutic proteins from blood plasma at point of care.

BACKGROUND OF THE INVENTION

Plasma products, such as fibrin sealants and plasma derived compositions, are widely used in numerous medical procedures. For example:

The Vivostat system for preparing autologous fibrin sealant - this system must be placed in the operating theater in order to withdraw autologous blood from a patient and can therefore be an open system as there is no actual“manufacturing” performed but only a“procedure” under the surgeon’s supervision. This system in no way allows or is compatible with producing the material at a manufacturing facility capable of producing sterile blood components (blood establishment, hospital blood bank etc.).

Purified stabilized therapeutic compositions. These include a variety of recombinant or plasma derived compositions that, however, all require to be quarantined for at least six months (blood components) or produced at a different manufacturing facility than point of care (stable plasma derived products [PDMPs] or recombinant proteins) and therefore require stabilization, lyophilization, freezing, and/or the addition of excipients most typically polymers or monomers of sugar moieties (e.g. mannitol, sucrose), but also human proteins (e.g. albumin), protease inhibitors (e.g. benzamidine, aprotinin).

Platelet Rich Plasma production systems: these systems only manipulate the cellular component utilizing physical filters and/or centrifugation and/or gels that are based on physical properties of density of the separated platelets. There is no electrostatic separation in these cases, specifically not at a molecular level of proteins. Also, these are not“closed” systems as the blood collection is always autologous and utilized at point of care on the same patient. Thus the resulting material is not a drug substance, but the whole system is used as part of a“procedure” and not as a manufacturing system. Blood component separation systems: these systems are routinely used in the collection and crude separation of blood into its cellular components (e.g. red blood cells and platelets) and non-cellular components (plasma) for therapeutic use and are indeed closed systems. However, these rely on purely physical separation methods - exclusively filtration or centrifugation, and do not allow for the integration of a biochemical separation module.

Therapeutic plasma exchange systems: these systems integrate a biochemical separation module (e.g. for LDL removal, CRP removal, therapeutic plasma exchange), but are used only in a therapeutic setting in continuous connection to the patient in order to filter components out of the blood, and cannot be used to produce or purify proteins therefrom.

There remains therefore an unfulfilled need for a production system capable of rapidly producing therapeutic proteins at blood establishments in a closed, sterile manner, from stored blood plasma units.

SUMMARY OF INVENTION

In one aspect, the present invention provides a sterile closed-system for producing a purified, enriched, therapeutic blood product, the system comprising: a sterile chromatography system comprising: (i) at least one separation module having fluid entrance and exit; (ii) at least one sterile container comprising or designed to contain or for holding sterile wash solution fluidly connected to said fluid entrance of said separation module; (iii) at least one sterile container comprising or designed to contain or for holding sterile elution solution fluidly connected to said fluid entrance of said separation module; (iv) at least one sterile collecting container, optionally comprising sterile dilution buffer, fluidly connected to said fluid exit of said separation module; (v) a waste container and/or a waste exit fluidly connected to said fluid exit of said separation module; (vi) a sterile connector suitable for sterilely connecting to a sterile container comprising plasma; (vii) at least one stopcock; and (viii) at least one one-way valve placed between at least some of the containers, at least one separation module, sterile connector and stopcock, ensuring directional process flow between the above containers; and at least one separation module, sterile connector and stopcock. In another aspect, the present invention provides a method for producing a purified, enriched, therapeutic blood product, the method comprising: (a) sterilely connecting a first container comprising at least one unit of sterile blood plasma to the sterile chromatography system of any one of the embodiments of the above mentioned first aspect using a sterile connection device; (b) separating and enriching one or more therapeutic blood factors according to a predetermined program comprising loading the at least one unit of sterile blood plasma on one of the least one separation module by means of one of the at least one pump in combination with at least one one-way valve, thereby binding one or more therapeutic blood factors on the first separation module, washing the first separation module by transferring a first washing buffer from one of the least one sterile container comprising washing buffer onto the separation module by means of one of the at least one pump in combination with at least one one-way valve, and eluting a fraction from the separation module by transferring from one of the least one sterile container comprising elution buffer onto the separation module a first elution buffer, which optionally comprises an activating agent, by means of one of the at least one pump in combination with at least one one-way valve, wherein the first fraction comprises one or more therapeutic blood factors, and optionally loading the first fraction on a second separation module by means of one of the at least one pump in combination with at least one one-way valve, washing the second separation module with a second washing buffer by means of one of the at least one pump in combination with at least one one-way valve, and eluting a second fraction from the second separation module with a second elution buffer by means of one of the at least one pump in combination with at least one one-way valve, wherein the second fraction comprises one or more therapeutic blood factors, wherein the directionality of flow at each step is controlled by the at least one one-way valve; and (c) collecting the fraction comprising the one or more therapeutic blood factors in one of the at least one collecting containers and optionally freezing the fraction, thereby producing the purified, enriched, therapeutic blood product.

In another aspect, the present invention further provides a sterile modular kit comprised of at least the following modules: (a) Infusion lines or infusion extension lines as the input to the kit, and compatible with sterile connection devices for the connecting of blood plasma bags; (b) Infusion lines or infusion extension lines as the output of the kit, compatible with sterile connection devices for the connecting of collection bags, optionally pre-connected to a bag, syringe, or other compatible collection device that may already be filled with buffers e.g. for the dilution of the product or addition of excipient; (c) buffer vessels capable of being manipulated by the corresponding electromechanical device (either bags or syringes); (d) tubing, either flexible or not flexible (e.g. plastic connectors), to enable the process flow; (e) Stopcocks, check valves, one-way valves, or any combination thereof, to direct process flow from the various inputs (buffers, plasma) according to the processing program pre-programmed into the electromechanical device; (f) at least one separation modules is independently selected from a column, filter, membrane or manifold comprising a resin selected from an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin; (g) at least one buffer vessel containing within or designed to contain a washing solution comprising up to 200m M anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, preferably chloride, e.g. up to 150m M of its sodium salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, butyric), negatively charged amino acids (e.g. serine, lysine, histidine, glutamic acid, aspartic acid) or positively charged amino acids (e.g. lysine, arginine and histidine); (h) at least one buffer vessel containing within or designed to contain an elution solution comprising between 200-2000 mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, e.g. up to 500mM of its sodium salt or 25 mM of its calcium salt essentially without additional different chloride salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, and butyric acid), negatively charged amino acids (e.g. serine, histidine, glutamic acid, aspartic acid), and positively charged amino acids, (e.g. lysine, arginine and histidine) ; (i) Optionally, an additional purification module - column, filter, or manifold, containing within a cation exchanger of the groups CM, S, SM or SP1 (j) optionally, an additional purification module - column, filter, or manifold, containing within a resin bound to an affinity ligand such as an antibody, protein A, protein G, or specific binding protein; (j) Optionally, an additional purification module - column, filter, or manifold, containing within a resin without any functional groups such as sephadex, agarose, sepharose, or other resins without functional groups; (k) Optionally, a buffer vessel containing a solution of solvent and/or detergent; (1) Optionally, a collection vessel pre-connected to the outgoing infusion line or infusion line extension; (m) all of the kit components are (i) Pre-sterilized by irradiation or individually sterilized and assembled aseptically; (ii) Pre-assembled as a closed system with no open ports communicating with the external atmosphere, and those ports being compatible with sterile connection devices to guarantee seamless production of a sterile product without a requirement for a sterile environment; and comprised of biocompatible plastic, glass, and/or metal components.

BRIEF DESCRIPTION OF DRAWINGS

Figs. 1A-C show a sterile closed-system for producing a partially purified, partially enriched, therapeutic blood product. (A) Assembly of a minimal sterile chromatography system (1) to be manipulated by an external pump and servo motor to direct flow of liquids. Input plasma (16) is connected using a sterile connection device to a flexible input tube (17) that is provided sealed (e.g. with a cap) and leads into a one way valve (4) (or check valve or other engineering solution enabling directional flow). The flow direction is indicated by the direction of the arrow. A three-way connector (5) (e.g. a T-connector or a Y connector) connected to a buffer container (3) allows the application of variable buffer compositions such as wash or elution buffers. This entire unit (2) (one-way valve (4), three-way connector (5), and buffer vessel (3) can be repeated to allow multiple buffers for washing, eluting a certain protein(s) composition, and eluting additional protein(s) compositions. These units are then connected through tubing (6) (flexible or rigid) to the separation module (7) (e.g. a column filled with a separation resin as described herein). A stopcock (9) (or other valve capable of directing flow in one of several directions) is guided by means of a servo motor (or similar element capable of rotating the valve and setting the flow direction) to either the waste (11) or product (10) side. The product is directed to a distribution manifold (8) in order to dispense one or more therapeutic doses. (B) The minimal repeating unit (11) of the distribution manifold (8) is comprised of a stopcock (13) (or other valve capable of directing flow in one of several directions) connected to a collection vessel (12). In this example the valve is a 3-way stopcock (flow directions indicated by 3-way arrow). Flow of product can be directed e.g. to (10) additional collection containers or additional separation modules past collection containers A and B (arrow indicating product flow direction); (14) to collection container A (arrow indicating flow direction), or (15) to collection vessel B (arrow indicating flow direction), etc’. (C) - A sterile chromatography system (1) comprising the elements depicted in (A) and (B) can be manipulated externally via an electromechanical device comprising a pump such as a peristaltic pump (19) that can integrate on the flexible tubing and thereby push plasma through the system and optionally also withdraw solution from the buffer vessel (e.g. by adding an additional pump and placing it on the tubing after the vessel). Alternatively the pump can be a syringe pump (18) that utilizes direct pressure on the buffer vessel in the form of a syringe to drive the solution through the kit, and aspirate plasma (16) by pulling on the syringe. The force is exerted on the plunger externally without compromising the internal solution sterility. The machine would include a controller element (20) (e.g. a Printed Circuit Board (PCB)), and some form of output (21) (e.g. LED lights and buttons). A motorized rotating element (22) (e.g. a servo motor) can direct flow of the solution by rotating the stopcock either towards the waste (down) or the collection/distribution element (right).

Figs. 2A-B show the removal of significant portion of antibodies in serum. (A) Western Blot for antibodies in all fractions (Goat anti human polyvalent immunoglobulins alkaline phosphatase conjugated was acquired from Sigma (A3313). The heavy chain (50 kDa) but not the light chain is well detected using this antibody. (B) - Coommassie staining of all fractions. M = Marker, P = Input plasma, W = Wash, E = Elution. NOTE - plasma was diluted 3 times as much as the other fractions prior to loading, due to the high concentration of antibodies affecting the running of the gel. Arrows point to the band representing the Immunoglobulin heavy chain in both gels.

Fig. 3 shows a reduction of approximately 7-fold in anti-B titer per ml between input plasma and ESC concentrate.

Fig. 4 depicts a run profile of a single step purification of FVIIa and FX. The trace marked with an asterisk is the optical density (OD) at a wavelength of 220nm. The trace marked with two asterisks shows the concomitant drop in conductivity indicative with the change of the buffer in this fraction from wash to activating solution. FVIIa activity (activated FVII) was measured in the 20 ml collected of the activation solution that include the peak indicated by an arrow.

Fig. 5 shows a Factor IX calibration curve performed with calibrator plasma showing a linear relation between the log dilution of the plasma and the time to clot.

Figs. 6A-B show adhesion area and score in rats that have undergone extensive abdominal surgery. (A) average adhesion area in the rats in the different groups. (B) adhesion scores also including the adhesion grading (a measure of its tenacity and vascularity).

Fig. 7 shows Factor V recovery and stability in ESC/CCC. Frozen, shock freezing of the composition at temperatures below -60°C; Cold, 4°C; RT, room temperature after 24 hrs (black) or 48 hrs (white).

Fig. 8 depicts a graph showing Factor V levels in cold storage in the absence (solid line) or presence of a dosage of heparin sufficient to inhibit 1% (dashed line) or 5% (dotted line) of the coagulation factors (0.01 and 0.05 units/ml, respectively).

Fig. 9 shows Factor V levels in ESC after freezing at -30°C.

Figs. 10A-B deptict data showing statistically significant reduction (>80%) after application of Coagulation Complex Concentrate (CCC) of the present invention in both area and score of adhesions in different compartments (A) and as sum of all regions measured (B). Black bars, control (untreated animals); Grey bars, animals treated with CCC obtained using the system and methods of the present invention comprising FII, FV, FVII, FVIII, FIX and FX.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of various embodiments, reference is made to the accompanying drawings that form a part thereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

In one aspect, the present invention provides a sterile closed-system for producing a purified, enriched, therapeutic blood product, the system comprising: a sterile chromatography system comprising: (i) at least one separation module having fluid entrance and exit; (ii) at least one sterile container comprising or designed to contain or for holding sterile wash solution fluidly connected to said fluid entrance of said separation module; (iii) at least one sterile container comprising or designed to contain or for holding sterile elution solution fluidly connected to said fluid entrance of said separation module; (iv) at least one sterile collecting container, optionally comprising sterile dilution buffer, fluidly connected to said fluid exit of said separation module; (v) a waste container and/or a waste exit fluidly connected to said fluid exit of said separation module; (vi) a sterile connector suitable for sterilely connecting to a sterile container comprising plasma; (vii) at least one stopcock; and (viii) at least one one-way valve placed between at least some of the containers, at least one separation module, sterile connector and stopcock, ensuring directional process flow between the above containers; and at least one separation module, sterile connector and stopcock.

In certain embodiments, the blood product comprises one or more therapeutic blood factors, such as coagulation factors.

In certain embodiments, the sterile closed system further comprises at least one pump designed to independently transfer the plasma, sterile wash solution, and sterile elution solution from their containers onto the at least one separation module without directly contacting the plasma, sterile wash solution, or sterile elution solution.

In certain embodiments, the at least one pump is at least one syringe pump or at least one peristaltic pump.

In certain embodiments, the sterile closed system further comprises a control module designed to control the operation of said at least one pump.

In certain embodiments, the sterile closed system further comprises a display unit.

In certain embodiments, the sterile chromatography system 1 comprises at least one three-way connector, such as a T-connector or a Y-connector, for fluidly connecting the above containers; and at least one separation module, sterile connector and stopcock via tubing.

In certain embodiments, one one-way valve and one container connected via a three-way connector comprises a container unit, which may be fluidly connected with identical container units, wherein each container contains or is designed to contain an identical or different solution, such as a wash solution or an elution solution.

In certain embodiments, the sterile closed-system is designed to produce 1 to 12 therapeutic doses of the purified, enriched, therapeutic blood product.

In certain embodiments, each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising an anion exchange resin; a column, filter, membrane or manifold comprising a cation exchange resin; a column, filter, membrane or manifold comprising a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a column, filter, membrane or manifold comprising a gel filtration/size exclusion resin.

In certain embodiments, each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising a resin selected from an anion exchange resin and a gel filtration/size exclusion resin.

In certain embodiments, the anion exchange resin is selected from Diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary ammonium (Q); the cation exchange resin is selected from carboxymethyl (CM), sulpho (S), sulphomethyl (SM) or sulphopropyl (SP); and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose; preferably, the anion exchange resin is Q.

In certain embodiments, the sterile connector is suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RadioFrequency (RF)- reactive thermoplastic material (aka as dielectric welding or radiofrequency welding), optionally in accordance with IS 09001, Good Manufacturing Practice (GMP) for blood banks, or code of Federal Regulations (CFR) standards for blood hanks.

In certain embodiments, the at least one sterile container is at least one sterile, syringe, plastic bag, glass vial or plastic vial. In certain embodiments, the at least one sterile container is at least one disposable sterile container.

In certain embodiments, the at least one sterile container of (ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump.

In certain embodiments, the at least one sterile container, e.g. at least one syringe, holds a volume of 1-50 ml, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 25, 30, 35, 40, 45, or 50ml, preferably 13, 25 or 50 ml. In certain embodiments, the at least one container designed to contain or comprising wash solution is one syringe operationally connected to one of the at least one syringe pumps, and the at least one container designed to contain or comprising elution solution is one syringe operationally connected to one of the at least one syringe pumps.

In certain embodiments, the at least one sterile collecting container is at least two sterile collecting containers fluidly connected to said fluid exit of said separation module via a manifold, wherein each sterile collecting container is connected to the manifold via a tube and a stopcock. The tube may be an infusion extension tube or an RF- compliant connector.

In certain embodiments, the stopcock connects one of the at least one separation module and the waste container and controls flow to either one of them.

In certain embodiments, the sterile closed system of the invention further comprises a power supply unit.

The display unit of the sterile closed system of the invention may comprise Light Enhanced Displays (LEDs), lights, sound-emitting devices, microphone, screen, or any combination thereof, capable of receiving a starting command and projecting information on the process (ready, working, finished, error).

In certain embodiments, the sterile closed system further comprises at least one pump designed to transfer the plasma, sterile wash solution, and sterile elution solution from their containers onto the at least one separation module without directly contacting the plasma, sterile wash solution, or sterile elution solution; the sterile closed system further comprises a control module designed to control the operation of said at least one pump; the sterile closed system further comprises a display unit; each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin; the sterile connector is suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RF-reactive thermoplastic material; the at least one sterile container of (ii), (iii) or (iv) is a syringe; and the at least one pump is at least one syringe pump; and the stopcock connects one of the at least one separation module and the waste container and controls flow to either one of them. In particular embodiments, the at least one sterile collecting container is at least two sterile collecting containers fluidly connected to said fluid exit of said separation module via a manifold, wherein each sterile collecting container is connected to the manifold via a tube and a stopcock.

In certain particular embodiments, each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin; the at least one container designed to contain or comprising wash solution is one syringe operationally connected to one of the at least one syringe pumps, and the at least one container designed to contain or comprising elution solution is one syringe operationally connected to one of the at least one syringe pumps;.

In certain particular embodiments, the anion exchange resin is selected from DEAE, QAE, and Q; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose; preferably, the anion exchange resin is Q.

In more particular embodiments, the sterile closed system comprises a sterile connector suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RF -reactive thermoplastic material; a first syringe operationally connected to one of the at least one syringe pumps; the first syringe is connected via a one-way valve to a second syringe comprising washing buffer and operationally connected to one of the at least one syringe pumps; the second syringe is connected via a one-way valve to a third syringe comprising elution buffer and operationally connected to one of the at least one syringe pumps; the third syringe is connected via a one-way valve to a first separation module comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin and optionally to a fourth syringe comprising elution buffer and operationally connected to one of the at least one syringe pumps; the fourth syringe, if present, is connected via a one-way valve to the first separation module; and the first separation module is connected via the stopcock to the waste container and via the stopcock and a manifold to at least two collecting syringes, wherein each collecting syringe is connected to the manifold via a tube and a stopcock. Reference is now made to Figs. 1A-C schematically illustrating a sterile closed- system for producing a purified, enriched, therapeutic blood product. The system comprises a sterile chromatography system 1 schematically illustrated in Figs. 1A-B comprising at least one repeating container unit 2 comprising a container 3, e.g. a syringe, a one-way valve 4, and a three-way connector 5. The repeating container unit 2 may be connected to an identical repeating container unit via tubing 6 and each container 3 contains or is designed to contain an identical or different solution, such as a wash solution or an elution solution or is intended to serve as a collection container of the product. One repeating container unit is fluidly connected to a separation module 7, which is fluidly connected to a waste container or a collection unit 8 via a stopcock 9 having at least one exit for product collection 10 and at least one exit for waste 11. The collection unit 8 may comprise one or more repeating container units 11, each one comprising a collection container 12 and a stopcock 13 controlling the flow. Each stopcock may be turned independently to allow flow to one of the collection containers 14 or another collection container 15.

More than one separation module 7 may be fluidly connected to the repeating container units. The sterile chromatography system 1 is fluidly connected to a sterile container comprising plasma 16 via a sterile connector 17 suitable for sterilely connecting to the sterile container 16. At the other end, the sterile chromatography system 1 is fluidly connected to one or more collection containers. For example, as shown in Fig. IB, in case there are more than one collection container, they are connected via a distribution manifold 8 comprising a minimal repeating unit 11 comprising a stopcock 13 connected to a collection container 12.

Fig. 1C shows examples of integration of the sterile chromatography system 1 with at least one pump, either a syringe pump 18 or a peristaltic pump 19; a control module 20; and a display unit 21 including displays for input (button/ LED/ keyboard/ touchpad etc’) to form the closed system. The control module comprises a computer controller / PCB / chip or similar device that controls the operations of the at least one pump and optionally server motor(s) 22 controlling the stopcocks. In certain embodiments, the sterile chromatography system 1 of any one of the above embodiments is a disposable, single -use, system, wherein the at least one sterile container of (ii) or (iii) comprises a wash solution or an elution solution.

In another aspect, the present invention provides a method for producing a purified, enriched, therapeutic blood product, the method comprising: (a) sterilely connecting a first container comprising at least one unit of sterile blood plasma to the sterile chromatography system of any one of the embodiments of the above mentioned first aspect using a sterile connection device; (b) separating and enriching one or more therapeutic blood factors according to a predetermined program comprising loading the at least one unit of sterile blood plasma on one of the least one separation module by means of one of the at least one pump in combination with at least one one-way valve, thereby binding one or more therapeutic blood factors on the first separation module, washing the first separation module by transferring a first washing buffer from one of the least one sterile container comprising washing buffer onto the separation module by means of one of the at least one pump in combination with at least one one-way valve, and eluting a fraction from the separation module by transferring from one of the least one sterile container comprising elution buffer onto the separation module a first elution buffer, which optionally comprises an activating agent, by means of one of the at least one pump in combination with at least one one-way valve, wherein the first fraction comprises one or more therapeutic blood factors, and optionally loading the first fraction on a second separation module by means of one of the at least one pump in combination with at least one one-way valve, washing the second separation module with a second washing buffer by means of one of the at least one pump in combination with at least one one-way valve, and eluting a second fraction from the second separation module with a second elution buffer by means of one of the at least one pump in combination with at least one one-way valve, wherein the second fraction comprises one or more therapeutic blood factors, wherein the directionality of flow at each step is controlled by the at least one one-way valve; and (c) collecting the fraction comprising the one or more therapeutic blood factors in one of the at least one collecting containers and optionally freezing the fraction, thereby producing the purified, enriched, therapeutic blood product. In certain embodiments, step (b) comprises partially separating and enriching one or more therapeutic blood factors

In certain embodiments, the purified therapeutic blood product is a partially purified therapeutic blood product.

In certain embodiments, the enriched therapeutic blood product is a partially enriched therapeutic blood product.

In certain embodiments, the at least one units of sterile plasma is (a) autologous plasma; (b) allogeneic plasma from a single donor (also called Fresh Frozen Plasma (FFP)) that is pre-tested for pathogens; or (c) pooled allogeneic plasma that is pretested for pathogens and treated for viral reduction. As mentioned, the blood plasma may be pretreated for pathogen reduction by e.g. amotosaleen-based methods, methylene blue- based methods; solvent-detergent methods, UVC irradiation, etc.

In certain embodiments, the allogeneic plasma from a single donor and the pooled allogeneic plasma that are pretested for pathogens and treated for viral reduction by solvent-detergent treatment and optionally filter-sterilized, such as Octaplasma®, a viral-reduced plasma produced from multiple units of plasma and treated for pathogen and viral reduction.

To illustrate the process by way of a non-limiting example, a separation module may be used, which is a 7ml column with an anion exchange resin. Three syringes (1, 2, and 3) are included in this specific system containing 25, 50, and 50 ml of water, wash buffer, and elution buffer, respectively. The flow direction, flow rate, solution volume, time allowed for each step, column volumes, and residence time are outlined in Table 1.

The term "column volume" as used herein refers to the volume of the separation column. The term "residence time" as used herein refers to the time that is required for one column volume to pass through the system. This value is obtained by dividing the flow rate through the system by the column volume.

In certain embodiments, the method produces 1 to 12 therapeutic doses of the purified, enriched, therapeutic blood product.

In certain embodiments, the method produces 1 to 12 therapeutic doses of the purified, enriched, therapeutic blood product starting from one unit of input blood plasma.

The chromatography system of the present invention may be sterilely connected a sterile container comprising plasma by any method providing a sterile connection. For example, there are systems for sterile connection that have a closed septum system. In these systems there are two parts - the acceptor (which in this case would belong to the chromatography system) and the donor (which would belong to the sterile container comprising plasma). Then there is a cover that, when the two parts are clicked together covers the connection to guarantee sterility. When the system and the plasma container are connected, the donor part penetrates the septum by a mechanism such as a spring- loaded needle making the connection.

Another option would be to use standard luer lock connectors on the plasma container and chromatography system side, that are connected in a sterile environment (e.g. a sterile hood).

In certain embodiments, the sterile connection device is a sterile tubing welder and the step of sterilely connecting the first container to a sterile chromatography system without compromising sterility of the unit comprises welding a tube comprising RF- reactive thermoplastic materials connected to first container to a tube comprising RF- reactive thermoplastic materials connected to the chromatography system by the means of the sterile tubing welder.

In certain embodiments, each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin.

In certain embodiments, the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin.

In certain embodiments, the anion exchange resin is selected from DEAE, QAE, and Q; the cation exchange resin is selected from CM, S, SM or SP; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose; preferably the anion exchange resin is Q.

In certain embodiments, the at least one sterile container of (ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump. In certain embodiments, the at least one container comprising wash solution is one syringe operationally connected to one syringe pump, and the at least one container comprising elution solution is one syringe operationally connected to one syringe pump.

In certain embodiments, the wash solution comprises up to 200mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, preferably chloride, e.g. up to 150mM of its sodium salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, and butyric acid), negatively charged amino acids (e.g. serine, histidine, glutamic acid, aspartic acid), and positively charged amino acids, (e.g. lysine, arginine and histidine).

In certain embodiments, the wash buffer comprises 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mM anions, e.g. in the form of NaCl. In certain embodiments the wash buffer comprises up to 200 mM NaCl or up to 100 mM NaCl.

In particular embodiments, the washing buffer comprises up to 80mM NaCl or up to 150mM NaCl, preferably 75mM NaCl or 100m M NaCl; and arginine and citrate, such as 5-50 mM arginine and citrate, preferably 10m M arginine and I OmM citrate, at physiological pH, such as 7.2-7.5, e.g. 7.4.

In certain embodiments, the elution buffer comprises between 200-2000 mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, e.g. up to 500mM of its sodium salt or 25 mM of its calcium salt essentially without additional different chloride salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, and butyric acid), negatively charged amino acids (e.g. serine, histidine, glutamic acid, aspartic acid), and positively charged amino acids, (e.g. lysine, arginine and histidine).

In certain embodiments, the elution buffer comprises 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mM anions, e.g. in the form of NaCl. In certain embodiments, the elution buffer comprises 250-300 mM NaCl, 250-500 mM NaCl, 300-500 mM NaCl, or 500-2000 mM NaCl.

In particular embodiments, the elution buffer comprises between 380-500 mM NaCl or 25 mM CaCl₂ essentially without additional different chloride salt; and arginine and citrate, such as 5-50 mM arginine and citrate, preferably I OmM arginine and I OmM citrate, at physiological pH, such as 7.4.

The sterile closed-system as defined herein may be used for producing any purified, enriched, therapeutic blood product, depending on the combination of type of separation module and wash and elution buffers used. For example, Table 2 discloses a number of coagulation factors and combinations thereof that can be produced with the sterile closed-system of the present invention using an anion exchange resin and exemplary conditions used. When a ligand column, such as a protein A or protein G column, is used to prepare a fraction of immunoglobulins, any physiological pH buffer may be used has wash buffer and any physiological buffer having a pH of 2-4 may be used for elution. A heparin-sepharose column may be used to prepare Antithrombin III typically along with a low salt wash buffer and high salt elution buffer.

In certain embodiments, the at least one unit of sterile, blood plasma, is (a) autologous plasma; (b) allogeneic plasma from a single donor that is pre-tested for pathogens; or (c) pooled allogeneic plasma that is pretested for pathogens and treated for viral reduction; the sterile connection device is a sterile tubing welder and the step of connecting the first container to a sterile chromatography system without compromising sterility of the blood plasma or chromatography system comprises welding a tube comprising RF -reactive thermoplastic materials connected to the first container to a tube comprising RF-reactive thermoplastic materials connected to the chromatography system by the means of a sterile tubing welder; each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin; the at least one sterile container of (ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump; the wash solution comprises up to 200mManions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, preferably chloride, e.g. up to 150m M of its sodium salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, and butyric acid), negatively charged amino acids (e.g. serine, histidine, glutamic acid, aspartic acid), and positively charged amino acids, (e.g. lysine, arginine and histidine); and the elution buffer comprises between 200-2000 mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate, e.g. up to 450mM of its sodium salt or 25 mM of its calcium salt essentially without additional different chloride salt; and a biocompatible buffer such as aliphatic acids (e.g. acetic, propionic, and butyric acid), negatively charged amino acids (e.g. serine, histidine, glutamic acid, aspartic acid), and positively charged amino acids, (e.g. lysine, arginine and histidine).

In certain particular embodiments, each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin; the at least one container comprising wash solution is one syringe operationally connected to one pump, and the at least one container comprising elution solution is one syringe operationally connected to one pump; the washing buffer comprises up to 80mM NaCl or up to 150m M NaCl, preferably 75mM NaCl or 100m M NaCl; and arginine and citrate, such as 5-50 mM arginine and citrate, preferably I OrnM arginine and I Orn M citrate, at physiological pH, such as 7.4; and the elution buffer comprises between 380-500 mM NaCl or 25 mM CaCF essentially without additional different chloride salt; and arginine and citrate, such as 5-50 mM arginine and citrate, preferably I OrnM arginine and I OrnM citrate, at physiological pH, such as 7.4.

In certain particular embodiments, the anion exchange resin is selected from DEAE, QAE, and Q; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose.

In certain particular embodiments, the anion exchange resin is Q.

For example, in order to partially purify and enrich Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X, the predetermined program comprises loading the blood plasma, such as a single donor allogeneic plasma pretested for pathogens, on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one way valve, washing the column by transferring a washing buffer comprising 100m M NaCl, I Orn M arginine and I Orn M citrate, at physiological pH, such as 7.4, by means of asyringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, eluting a fraction from the anion exchange column by

transferring an elution buffer comprising 420mM NaCl, I Om M arginine and I Om M citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises Factor II, Factor V, Factor VII Factor VIII, Factor IX, and Factor X, thereby producing the single therapeutic dose of a therapeutic blood product comprising non- stabilized, partially purified, partially enriched Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X. In this example, the input plasma was also tested, and the composition generated is compared to the input plasma. Since the volume is reduced approximately l2-fold during the procedure, the factors are effectively concentrated. Two numbers can be obtained from this calculation method. One number is the concentration factor - how much factor activity per ml is found in the extract compared to the input plasma. The second number is the recovery of activity, i.e. the concentration factor divided by the volumetric reduction (12-fold). These numbers are shown in Table 3: Some factors (e.g. factors VIII and IX) were tested in a different experiment (see example 5) where a slightly different calculation method was used but with similar outcomes.

In certain embodiments, this method results in a recovery efficiency of Factor II of at least 60, 65, 70, 75, 80, 85, 90, or 95%.

In certain embodiments, this method results in a recovery efficiency of Factor II of 60-100%, 70-90%, 70-100, 80-90, 80-100, 90-100, or 80-85%. In certain embodiments, this method results in a recovery efficiency of Factor II of 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

In certain embodiments, this method results in a concentration factor of Factor II of at least 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200%.

In certain embodiments, this method results in a concentration factor of Factor II of 800-1200%, 800-1100%, or 900-1100%.

In certain embodiments, this method results in a concentration factor of Factor II of 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200%.

In certain embodiments, this method results in a recovery efficiency of Factor V of at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%.

In certain embodiments, this method results in a recovery efficiency of Factor V of 25-50%, 30-45%, 30-40% or 30-35%.

In certain embodiments, this method results in a recovery efficiency of Factor V of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%.

In certain embodiments, this method results in a concentration factor of Factor V of at least 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain embodiments, this method results in a concentration factor of Factor V of 300-1000%, 350-600%, 400-500% or 400-450%.

In certain embodiments, this method results in a concentration factor of Factor V of 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain embodiments, this method results in a recovery efficiency of Factor VII of at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

In certain embodiments, this method results in a recovery efficiency of Factor VII of 40-100%, 50-80, 50-90, 50-100, 60-80%, 60-90, 60-100%.

In certain embodiments, this method results in a recovery efficiency of Factor VII of 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In certain embodiments, this method results in a concentration factor of Factor VII of at least 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400%.

In certain embodiments, this method results in a concentration factor of Factor VII of 800-1400%, 800-1300, 900-1400, 900-1300, 1000-1400, 1000-1300, 1100-1400, 1100-1300, 1200-1400, or 1200-1300%.

In certain embodiments, this method results in a concentration factor of Factor VII of 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400%.

In certain embodiments, this method results in a recovery efficiency of Factor X of at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

In certain embodiments, this method results in a recovery efficiency of Factor X of 40-100%, 50-80, 50-90, 50-100, 60-80%, 60-90, 60-100%.

In certain embodiments, this method results in a recovery efficiency of Factor X of 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

In certain embodiments, this method results in a concentration factor of Factor X of at least 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400%.

In certain embodiments, this method results in a concentration factor of Factor X of 800-1400%, 800-1300, 900-1400, 900-1300, 1000-1400, 1000-1300, 1100-1400, 1100-1300, 1200-1400, or 1200-1300%.

In certain embodiments, this method results in a concentration factor of Factor X of 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400%.

In order to partially purify and enrich Factor V, the fraction comprising Factor II, Factor V, Factor VII and Factor X may be loaded on a column comprising the gel filtration/size exclusion resin sephadex, eluting the column with an elution buffer comprising physiological concentrations of buffered sodium chloride, the buffer being a physiologically acceptable buffer, such as citrate, arginine, lysine and/or histidine, and collecting a first fraction in one of the at least one collecting container, wherein the first fraction comprises Factor V, thereby producing the single therapeutic dose of a therapeutic blood product comprising non-stabilized, partially purified, partially enriched Factor V; and collecting a second fraction in one of the at least one collecting container, wherein the second fraction comprises Factor II, Factor VII and Factor X thereby producing the single therapeutic dose of a therapeutic blood product comprising non- stabilized, partially purified, partially enriched Factor II, Factor VII and Factor X.

In certain embodiments, in order to partially purify and enrich Factor VII, the predetermined program comprises loading the blood plasma, such as a single donor allogeneic plasma pretested for pathogens, on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, washing the column by transferring a washing buffer comprising 100m M NaCl, 10m M arginine and lOmM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, eluting a fraction from the anion exchange column by transferring an elution buffer comprising 300mM NaCl, lOmM arginine and lOmM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in the first of the at least one collecting container, wherein the fraction comprises partially purified and partially enriched Factor VII thereby producing the single therapeutic dose of a therapeutic blood product comprising a partially purified, partially enriched Factor VII.

In certain particular embodiments, this method results in a recovery efficiency of Factor VII of at least 15%, preferably at least 40%, a concentration factor of at least 200%, preferably at least 500%, and a relative purity of at least 80%.

In certain particular embodiments, this method results in a recovery efficiency of Factor VII of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or

50%.

In certain particular embodiments, this method results in a recovery efficiency of Factor VII of 10-50%, 15-45%, or 17-44%.

In certain particular embodiments, this method results in a recovery efficiency of Factor VII of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% In certain particular embodiments, this method results in a concentration factor of Factor VII of at least 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, this method results in a concentration factor of Factor VII of 150-1000, 150-900, 150-800, 150-700, 150-600, 150-500, 150-400, 150- 300, 150-200, 300-1000, 300-900, 300-800, 6300-700, 300-600, 300-600, 300-500, 300- 400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500- 800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700- 800, 800-1000, 800-900, or 900-1000%.

In certain particular embodiments, this method results in a concentration factor of Factor VII of 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, this method results in a relative purity of Factor VII of at least 70, 75, 80, 85, 90, or 95%.

In certain particular embodiments, this method results in a relative purity of Factor VII of 70-100, 70-90, 70-80, 80-100, 80-90, 90-100, or 90-95%.

In certain particular embodiments, this method results in a relative purity of Factor VII of 70, 75, 80, 85, 90, or 95%.

The term relative purity of a coagulation factor as used herein refers to the percent recovery of the activity of the factor compared to the recovery of all of the extrinsic factors (namely factors II, VII, V, and X) vs. the respective activity in the input plasma.

In certain embodiments, in order to partially purify and enrich Factor Vila, the predetermined program comprises loading the blood plasma, such as a single donor allogeneic plasma pretested for pathogens, on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, washing the column by transferring a washing buffer comprising 75mM NaCl, I OmM arginine and I OmM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, activating and eluting a bound fraction on the anion exchange column by transferring a buffer comprising at least 15mM CaCl₂, preferably 25mM CaCl₂, and 10m M arginine and 10m M citrate, at physiological pH, such as 7.4, essentially without additional different chloride salt, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises partially purified and partially enriched Factor Vila thereby producing the single therapeutic dose of a therapeutic blood product comprising a partially purified, partially enriched Factor Vila.

In certain particular embodiments, this method results in a recovery efficiency of Factor Vila of at least 50%, preferably at least 65%, a concentration factor of at least 300%, preferably at least 400%, and a relative purity of at least 80%, preferably at least 95%.

In certain embodiments, this method results in a recovery efficiency of Factor Vila of at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

In certain embodiments, this method results in a recovery efficiency of Factor Vila of 40-100%, 50-80, 50-90, 50-100, 60-80%, 60-90, 60-100%.

In certain embodiments, this method results in a recovery efficiency of Factor Vila of 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

In certain embodiments, this method results in a concentration factor of Factor Vila of at least 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%.

In certain embodiments, this method results in a concentration factor of Factor Vila of 200-500, 200-400, 200-300, 300-500, or 300-400%.

In certain embodiments, this method results in a concentration factor of Factor Vila of 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%.

In certain particular embodiments, this method results in a relative purity of Factor Vila of at least 70, 75, 80, 85, 90, or 95%.

In certain particular embodiments, this method results in a relative purity of Factor Vila of 70-100, 70-90, 70-80, 80-100, 80-90, 90-100, or 90-95%. In certain particular embodiments, this method results in a relative purity of Factor Vila of 70, 75, 80, 85, 90, or 95%.

It is possible to elute one or two additional factors from the resin already eluted with the CaCF-containing elution buffer by eluting with e.g. a NaCl containing buffer.

Thus, in certain embodiments, in order to partially purify and enrich Factor X, the method further comprises the step of eluting a second fraction from the column with an elution buffer comprising about 300mM NaCl and 10m M arginine and 10m M citrate, at physiological pH, such as 7.4 by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises partially purified and partially enriched Factor X, thereby producing the single therapeutic dose of a therapeutic blood product comprising a partially purified, partially enriched Factor X.

In certain particular embodiments, this method results in a recovery efficiency of Factor X of at least 20%, preferably at least 25%, a concentration factor of at least 100%, preferably at least 150%, and a relative purity of at least 80%, preferably at least 85%.

In certain particular embodiments, this method results in a recovery efficiency of Factor X of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%.

In certain particular embodiments, this method results in a recovery efficiency of Factor X of 10-50%, 10-40, 10-30, 10-20, 20-50, 20-40, 20-30, 30-50, 30-40, 40-50, or 20-25%.

In certain particular embodiments, this method results in a recovery efficiency of Factor X of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50%

In certain particular embodiments, this method results in a concentration factor of Factor X of at least 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300%.

In certain particular embodiments, this method results in a concentration factor of Factor X of 75-300, 80-300, 80-275, 80-250, 80-225, 80-200, 80-175, 80-150, 80-125, 80-100, 100-300, 100-275, 100-250, 100-225, 100-200, 100-175, 100-150, or 100-125% In certain particular embodiments, this method results in a concentration factor of Factor X of 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300%.

In certain particular embodiments, this method results in a relative purity of Factor X of at least 70, 75, 80, 85, 90, or 95%.

In certain particular embodiments, this method results in a relative purity of Factor X of 70-100, 70-90, 70-80, 80-100, 80-90, 90-100, or 90-95%.

In certain particular embodiments, this method results in a relative purity of Factor X of 70, 75, 80, 85, 90, or 95%.

In certain alternative embodiments, in order to partially purify and enrich Factor X and Factor II, the method further comprises the step of eluting a second fraction from the column with an elution buffer comprising about 420m M NaCl and 10m M arginine and I OmM citrate, at physiological pH, such as 7.4 by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises partially purified and partially enriched Factor X and Factor II, thereby producing the single therapeutic dose of a therapeutic blood product comprising a partially purified, partially enriched Factor X and Factor II.

In certain particular embodiments, this method results in a recovery efficiency of Factor X of at least 50%, preferably at least 70%, a concentration factor of at least 100%, preferably at least 450%, and a relative purity of at least 80%, preferably at least 95%.

In certain embodiments, this method results in a concentration factor of Factor X of at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%. In certain embodiments, this method results in a concentration factor of Factor X of 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, or 300- 400%.

In certain embodiments, this method results in a concentration factor of Factor X of 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%.

In certain particular embodiments, this method results in a recovery efficiency of Factor II of at least 50%, a concentration factor of at least 300%.

In certain embodiments, this method results in a recovery efficiency of Factor II of at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

In certain embodiments, this method results in a recovery efficiency of Factor II of 40-100%, 50-80, 50-90, 50-100, 60-80%, 60-90, 60-100%.

In certain embodiments, this method results in a recovery efficiency of Factor II of 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

In certain embodiments, this method results in a concentration factor of Factor II of at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%.

In certain embodiments, this method results in a concentration factor of Factor II of 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, or 300- 400%.

In certain embodiments, this method results in a concentration factor of Factor II of 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500%.

In certain particular embodiments, this method results in a relative purity of FII of at least 45, 50, 55, 60, 65, or 70%. In certain particular embodiments, this method results in a relative purity of the combined FX+FII product vs. other factors of the Extrinsic System (FVII and FV) of at least 97%.

In certain particular embodiments, this method results in a relative purity of the combined FX+FII product of at least 70, 75, 80, 85, 90, 95, or 97%.

In certain particular embodiments, this method results in a relative purity of the combined FX+FII product of 70-100, 70-97, 70-90, 70-80, 80-100, 80-97, 80-90, 90-100, 90-97, or 90-95%.

In certain particular embodiments, this method results in a relative purity of the combined FX+FII product of 70, 75, 80, 85, 90, 95, or 97%.

Alternatively, the method comprises first eluting a first fraction of partially purified, partially enriched Factor X and Factor II with an elution buffer comprising 300 mM NaCl and then eluting a second fraction of partially purified, partially enriched Factor X and Factor II with an elution buffer comprising 420 mM NaCl, and combining the first and the second fraction.

In certain embodiments, the blood product is practically devoid of hemoagglutinins, i.e. the blood product contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of the level of hemoagglutinins in the input plasma.

In certain embodiments, the concentration factor of Factor II is at least 200%, the concentration factor of Factor V is at least 100%, the concentration factor of Factor VII is at least 200%, the concentration factor of Factor VIII is at least 100%, the concentration factor of Factor IX is at least 200%, or the concentration factor of Factor X is at least 200%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor II, Factor IX and Factor X of at least 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor II, Factor IX and Factor X of 150-1000, 150-900, 150-800, 150- 700, 150-600, 150-500, 150-400, 150-300, 150-200, 300-1000, 300-900, 300-800, 6300- 700, 300-600, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400- 600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600- 800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor II, Factor IX and Factor X of 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750,

775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor V and Factor VIII of at least 75, 100, 125, 150, 175, 200, 225, 250,

275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700,

725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor V and Factor VIII of 100-1000, 100-900, 100-800, 100-700, 100- 600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200- 600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 6300-700, 300-600, 300- 600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500- 1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700- 1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000%.

In certain particular embodiments, this method results in a concentration factor of each one of Factor V and Factor VIII of 150, 175, 200, 225, 250, 275, 300, 325, 350,

375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800,

825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain embodiments, the blood product produced according to any one of the above embodiments is a labile and non-stabilized blood product.

In certain embodiments, the method of any one of the above embodiments further comprises stabilizing the blood product by adding a cryoprotectant, such as sucrose, mannitol, trehalose, polyethyleneglycol (PEG) at various molecular weights (e.g. 400, 600, 3350 g/mol), and glycine; or an anticoagulant, such as heparin or a derivative thereof and enoxaparin sodium, and storing it, thereby producing a stabilized blood product. In certain embodiments, the cryoprotectant is sucrose at a final concentration of about 2% (w/v) and the method further comprising freezing the stabilized blood product by exposing it to a temperature of -30°C and subsequently storing it at about -30°C.

In certain embodiments, the blood product, after storing up to about four weeks or longer, comprises Factor V having a level of activity that is essentially equal to freshly produced Factor V.

In certain embodiments, the anticoagulant is heparin at a final concentration of about 1% (w/v); and the method further comprises storing the stabilized blood product at about 2-8°C.

In certain embodiments, the blood product, after storing up to about four weeks or longer, comprises Factor V having a level of activity that is about 25% of that of freshly produced Factor V, respectively.

In certain embodiments, the composition is practically or essentially devoid of hemoagglutinins .

In a further aspect, the present invention provides a pharmaceutical composition obtained by any one of the embodiments of the method for producing a partially purified, partially enriched, therapeutic blood product of the present invention comprising, or essentially consisting of, any combination of non-stabilized, partially purified, partially enriched Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X and a pharmaceutically acceptable carrier.

In certain embodiments the above pharmaceutical composition essentially consists of non-stabilized or stabilized, partially purified, partially enriched: (a) Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X; (b) Factor II, Factor V, Factor VII, and Factor X; (c) Factor II, Factor V, Factor VII, Factor VIII, and Factor X; (d) Factor II, Factor V, Factor VII, Factor IX, and Factor X; (e) FVII as a sole blood factor; (f) FVIIa as a sole blood factor; (g) FX as a sole blood factor; or (h) FII and FX as the sole blood factors; and a pharmaceutically acceptable carrier.

In certain particular embodiments, the concentration factor of each one of Factor II, Factor IX and Factor X is at least 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, the concentration factor of each one of Factor II, Factor IX and Factor X is 150-1000, 150-900, 150-800, 150-700, 150-600, 150-500, 150-400, 150-300, 150-200, 300-1000, 300-900, 300-800, 6300-700, 300-600, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000%.

In certain particular embodiments, the concentration factor of each one of Factor II, Factor IX and Factor X is 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, the concentration factor of each one of Factor

V and Factor VIII is at least 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%.

In certain particular embodiments, the concentration factor of each one of Factor V and Factor VIII is 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 6300-700, 300-600, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-800, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000%.

V and Factor VIII is 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, or 1000%. In certain embodiments, the pharmaceutical composition further comprises a cryoprotectant, such as sucrose, mannitol, trehalose, polyethyleneglycol (PEG) at various molecular weights (e.g. 400, 600, 3350 g/mol), and glycine; or an anticoagulant, such as heparin or a derivative thereof and enoxaparin sodium, i.e. the pharmaceutical composition comprises a stabilized blood product.

In certain embodiments, the cryoprotectant is sucrose at a final concentration of about 2% (w/v).

In certain embodiments, the blood product, after freezing the pharmaceutical composition by exposing it to a temperature of -30°C and subsequently storing it at about -30°C up to about four weeks or longer, comprises Factor II or Factor V having a level of activity that is essentially equal to freshly produced Factor II or Factor V, respectively.

In certain embodiments, the anticoagulant is heparin at a final concentration of about 1% (w/v.

In certain embodiments, the blood product, after storing it at 2-8°C up to about four weeks or longer, comprises Factor V having a level of activity that is about 25% of that of freshly produced Factor V, respectively.

In certain embodiments, the composition of any one of the above embodiments is practically devoid of hemoagglutinins.

In certain embodiments, the solvent and/or detergent in any one of the embodiments of the present invention is a nonionic detergent/surfactant, such as Polysorbate 20 (e.g. Tween®-20 or Tween® 80), a nonionic surfactant that has a hydrophilic polyethylene oxide chain and a 4-phenyl hydrocarbon group of the formula wherein n=9-l0 (e.g. Triton X-100®),

nonylphenoxypolyethoxylethanol (e.g. NP-40), or tri(n-butyl)phosphate.

In conclusion and in summary of these experiments, the present invention provides for the utilization of blood bank technology, i.e. the use of quarantined or pre treated plasma units available in blood banks and ready to be applied directly to patients, together with a closed system ready to be utilized within the blood bank without breaking sterility at any point.

This novel system can:

A. Be used in a blood bank or point-of-care setting and not in a manufacturing facility.

B. Be used to generate a single therapeutic dose (or less than 12).

C. Be used to generate any of the therapeutic coagulation factor proteins including the currently unavailable as a stable protein, Factor V (A.K.A. pro-accelerin, FV, or labile factor).

D. Be used to generate any combination of coagulation factors, most notably FII, FV, FVII, FVIII, FIX and FX but also FII, FV, FVII, and FX (ESC), and others.

E. Be used to generate a minimally or partially purified concentrated therapeutic protein or composition thereof that can be used directly on patients due to the method of production .

F. Can be used to prepare any of the above compositions rapidly, under 6 hours, 4 hours, 2 hours, or even as little as 1 hour or less, as a final composition.

G. Utilizing blood bank compatibility, rely on the blood bank supplied input plasma to be safe (tested/quarantined/pathogen inactivated/screened etc’).

H. Be used to prepare any of the compositions above with no requirement for stabilizing excipients if applied within several days.

I. Be used to prepare a stable factor V containing ESC or CCC by at -30°C without exposure to lower temperatures. J. Utilizing blood bank input plasma as safe plasma, be used to prepare short lived (<6 months = quarantine time of plasma products) products, or as little as one month, one week, or even 1-2 days.

K. Prepare blood group type independent therapeutic proteins based on its capacity to reduce the volume and titer of hemoagglutinin antibodies by at least 10 fold, preferably even as much as 40-50 fold or even 100 fold or more (depending on the wash conditions).

For purposes of clarity, and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values recited herein, should be interpreted as being preceded in all instances by the term "about." Accordingly, the numerical parameters recited in the present specification are approximations that may vary depending on the desired outcome. For example, each numerical parameter may be construed in light of the number of reported significant digits and by applying ordinary rounding techniques, or it may mean that values which are 10% above or below the value provided are also included.

The term "therapeutic dose" as used herein refers to an amount of material for the treatment of one patient sufficient to cover an area affected by surgery, e.g. about 5 ml or more of a therapeutic blood product as defined herein, or a volume sufficient to cover an area equivalent to approximately three sheets of SEPRAFILM® (Sanofi Biosurgery), a medical device for adhesion prevention for patients undergoing abdominal or pelvic laparotomy.

The term "partially purified" as used herein refers to the relative abundance of a therapeutic blood factor relative to other blood plasma proteins (aka relative purity), which is higher than in the source blood plasma but lower than in pure (100%) therapeutic blood factor, i.e. the blood product is not essentially free of other proteins (e.g other blood plasma proteins such as immunoglobulins, or other coagulation factors).

The term "partially enriched" as used herein refers to the relative concentration of a therapeutic blood factor as measured e.g. by its activity per volume, which is higher than in the source blood plasma. The protein is not typically concentrated to a very high level such as can be found in standard pharmaceutical preparations (e.g. commercial Prothrombin Complex Concentrate [PCC] which is prepared at a concentration of 2000% compared to standard plasma levels; Fibrin sealants which contain up to 1000 units of highly purified thrombin per ml and up to 13% w/v or approximately 50-fold concentration of Fibrinogen compared to standard plasma levels).

The term "concentration factor" as used herein refers to the volume of input relative to the volume of output.

The term "fold activity" as used herein refers to the activity in the output relative to the activity in the input.

The term "recovery" as used herein refers to the fold activity over the concentration factor. For example, a method in which the“concentration factor” is 5 (e.g. 125 ml of plasma concentrated into 25 ml of extract: 125/25 = 5), and the activity is 4-times higher in the output/input (fold activity = 4-fold or 400%) yields a recovery of 4/5 (fold activity/concentration factor) or 80%.. .

The term "therapeutic blood product" as used herein refers to a product comprising one or more of blood factors II, V, VII, VIII, IX, and X or their activated forms.

The term "stopcock" as used herein refers to any kind of valve in a pipe or tube that controls the flow of liquid through it. The valve is capable of switching flow direction between two or more routes.

The terms "check valve" and "one-way valve" are used interchangeably herein and refer to any mechanical configuration that enables flow of liquid through tubing in only one direction when liquid pressure is applied. For example, check valves, directionally oriented septum components, mechanical gating elements where several “leaves” are oriented in an iris formation so that pressure in one direction will allow them to open but pressure in the other direction will push the“leaves” against each other thereby forcing their closure, or any other engineering solution to that end.

The term "sterile connector suitable for sterilely connecting to a sterile container comprising plasma" as used herein preferably refers to a device which integrates a sterile chamber, a cutting element, a heating element, and an alignment element. The device cuts the input and output tubes of the kit and plasma respectively in said chamber, aligns them, and heats them using RF transmission so as to weld the two tubes together in a sterile fashion The term "plasma pre-tested for pathogens" as used herein refers to plasma that is intended for therapeutic purposes such as infusion into patients and is therefore tested for the presence of known blood-borne pathogens such as hepatitis (HBV, HCV), HIV, etc’.

The term "pretreated for pathogen reduction with solvent-detergent" as used herein refers to plasma that undergoes an additional active step towards the further reduction of said pathogens that is based on the addition of solvents and detergents that dissolve the membrane surrounding these pathogens.

Plasma pre-treated for pathogens may also include plasma treated by filtration, amotosaleen treatment, UV-irradiation, solvent-detergent treatment, prion- ligand column treatment, etc’.

The term "blood plasma approved for transfusion in a blood establishment" as used herein refers to blood plasma which has successfully tested negative for known pathogens and is therefore stored in blood establishments such as blood hanks and is approved by the respective country ministries (e.g. Ministry of Health) for the transfusion into patients such as would require plasma transfusion.

The term "plasma" or "blood plasma" as used herein refers to the liquid part of blood.

The term“pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

The term "essentially devoid" is used interchangeably with "practically devoid" "substantially devoid", "essentially lacking" or "near absence" in the context of a composition, and refers to a concentration of a component of that composition not exceeding about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%.

The invention will now be illustrated by the following non-limiting examples. EXAMPLES

Example 1. Concentration of Extrinsic System Concentrate (ESC) using an automated system with a disposable kit

Laboratory testing of factor II, V, VII and X levels was performed using Prothrombin Time (PT)-based assays; and activated Partial Prothrombin Time (aPTT) based assay for factors VIII, and IX. In the standard aPTT assay phospholipids and calcium are added to plasma in order to activate clotting and the clotting time is recorded. In order to assay the activity of a certain coagulation enzyme such as factor IX or VIII in a composition, plasma that has been depleted of the respective factor is used, and the composition is added to complement the activity of this factor. By preparing a standard curve by complementing the factor depleted plasma with various dilutions of normal or international standard plasma, and comparing the clotting times to those obtained with various dilutions of the tested composition, the factor concentration can be interpolated.

The PT based assay is very similar to the aPTT based assay, except the activator of coagulation also includes Thromboplastin (a mixture containing Tissue Factor protein).

ESC is useful in adhesion prevention (our PCT). The FuseX system developed by EIO Biomedical was utilized. In this system, a blood infusion set was used to connect the system to input plasma in accordance with blood bank technology. The electromechanical device was then used to drive the plasma, wash, and elution buffers through the column, resulting in a final collection syringe (Fig. 1).

The final collection syringe can be replaced with a manifold via which at least two or five syringes or any number of syringes can be connected. The manifold and each one of the collecting syringes is connected to the system via extension tube and enabling sterile disconnection (Fig. 1).

The protocol included loading approximately 125 ml of plasma on a single use column containing 5 ml of anion exchange resin (red cap in the figures above), a 100m M buffered NaCl wash, and approximately 400-450 mM NaCl elution. Extrinsic System Concentrate factors were tested for activity in the collection syringe and the following recoveries were seen:

A single unit of plasma is capable of producing as much as 30 ml of active material, sufficient for a large laparotomy. Thus, an automated system, compatible with sterile connections and using blood bank technology was used to generate a therapeutic dose of concentrated ESC that is useful for adhesions prevention.

Example 2. Blood type independence of generated product.

Surprisingly, our method of producing coagulation factors with a single step purification (single anion exchange column) is sufficient to significantly reduce the titer of hemoagglutinins. These are antibodies generated in the blood of many people with a certain blood group (e.g. ABO) against the antigen that is not present in their system. Thus, typically persons with blood group A will present antibodies against blood group B, and vice versa (blood group O will present antibodies against both antigens).

We performed three different wash conditions (50mM, lOOmM, and l50mM) prior to eluting the column, and performed both Western blotting and Coomassie protein detection for the antibodies in the eluate (Figs. 2A-B). All conditions showed the removal of the significant portion of antibodies in the wash, with much lower levels of antibody remaining in the elution compared to the input plasma (NOTE that plasma samples were diluted 3 times as much as the wash and elution samples in order to enable proper gel running). In addition to the visible reduction in antibody titer, there was also a quantitative reduction in the level of hemoagglutinin anti-B antibodies in extracts prepared from blood group A type plasma.

Quantitative analysis of anti- blood group B titer from five independent runs in the system also show a reduction of approximately 7-fold in the titer per ml between input plasma and ESC concentrate (Fig. 3). Considering that the volume reduction was approximately 6-fold in the ESC, that translates to an approximately 44-fold reduction in the titer of hemoagglutinins in the product of the system. Thus, the system can be used to prepare a therapeutic protein that is blood group type independent, originating from as little as one blood unit.

In conclusion, a single step purification using blood bank technology is sufficient to generate a therapeutic dose of a highly concentrated protein mix with sufficient reduction of hemoagglutinins to render the product blood group type independent.

Example 3. Purification of FVII using a single disposable column (single step) chromatography.

FVII was purified and concentrated from other factors tested (FII, FV, and FX) in a single step purification.

Approximately 125 ml plasma were loaded on a miniaturized column pre-loaded with 5 ml of anion exchange resin (Q). Following a washing step of a buffered 100m M NaCl solution, isocratic elution steps were performed to yield several compositions of partially purified and concentrated FVII. Factor activity was tested in the different elution steps.

Results:

* concentration factor of factor over input plasma is calculated by multiplying the recovery by the volumetric concentration factor of the elution vs. the input plasma. In this case about 12-13 fold.

An alternative Protocol:

Loading 125 ml plasma on an anion exchange column (5ml Q fast flow [GE])

Washing with citrate/arginine buffered NaCl 100 mM

1. Washing with citrate/arginine buffered NaCl 200mM

2. Eluting with citrate/arginine buffered NaCl 250mM

3. Eluting with citrate/arginine buffered NaCl 300mM

4. Freezing the material and thawing it for testing

Factor VII was separated completely from other PPSB factors (e.g. FII, FX), and separated from the majority of activity of the co-factor V. Factor V can be easily reduced, however, by freeze-thawing the sample. In this example, the activity of FV was reduced to 0.6% and 1.8% from 1% and 8%, in the 250mM and 300mM elutions, respectively. This increased the purification factor of the FVII activity to >95% from the other enzymatic coagulation factors.

In terms of yield, if a single step of 300mM were used, it can be easily calculated that the recovery in one step would be as high as 61% which (in this example) would be equivalent to over 7.5-fold concentration compared to input plasma.

The normal Factor VII plasma concentration is 0.5 pg/mL. Factor VII levels of 15-25% (0.075 - 0.125 pg/mL) are generally sufficient to achieve normal hemostasis (Bauer, K.A.: Treatment of Factor VII deficiency with recombinant Factor Vila, Haemostasis 1996; 26 (suppl 1): 155-158.). Recombinant FVIIa is only recovered at 20% activity in plasma (NovoSeven® Instructions For Use).

As the maximal blood plasma volume of a 70 kg man is approximately 3000 ml, a simple calculation would show that to restore 15% of that activity, using this example, about 750 ml of plasma would need to be processed. This is less than 4 units, which can be pooled using available blood bank technology.

Thus, a single therapeutic dose of partially purified FVII concentrate can be produced using our system.

Example 4. PURIFICATION OF FVIIa and FX using a single anion exchange column (single step) chromatography

FVII can be activated by Calcium Chloride in the presence of other coagulation factors (e.g. Blood, Vol. 60, No. 5 (November), 1982).

We performed a single step (single column) protocol to produce FVIIa.

125 ml were loaded on a miniature disposable column preloaded with 5 ml of anion exchange resin as described in the first example. Following a 75mM buffered NaCl wash, a 25mM Calcium chloride activation solution was loaded at a rate of 0.5ml/min. The first 20 ml of this activation solution yielded a sharp peak (Fig. 4), and this was collected and tested for FVII activity. Flla (activated factor II, or Thrombin) was not present in this fraction (tested using an assay for thrombin activity). FVIIa, on the other hand, was highly concentrated in this fraction. 68% of FVII activity was recovered in this fraction, thus indicating that not only can we produce FVII separated from other coagulation factors, but also generate active FVIIa in a single column (single step) purification.

In order to isolate Factor X (FX), we performed a buffered NaCl elution in two steps, 300 and 420mM. Approximately 26% of FX was isolated in the 300mM elution step, without any traces of FVII (as this has already been collected as described in this example). FII and FV levels were approximately 2.7% and 1.1%, respectively, thus FX activity was >85% in this single separation step.

* FVII can be activated by Calcium Chloride in the presence of other coagulation factors (e.g. Blood, Vol. 60, No. 5 (November), 1982).

** To make this product a single 420mM elution would be used and this number was used for the calculations

*** Calculated for the combined product: FX and FII

FII and the residual FX activity were detected in the 420mM elution step (an additional 50% of FII activity, and 48% of FX activity). Thus, by prolonging the 300mM elution step, it would be straightforward to obtain a largely separated FX, and thereby also a largely separated FII fraction. In this protocol FV is activated via CaCF and is therefore very short-lived, and FV activity would not contaminate the preparation.

It is also quite clear from our examples here, that it is possible to also obtain novel mixtures of factors II, V, VII and X. For example, in the 420m M elution we were able to get a large proportion of FII and FX (approximately 50%) with less than 2% of FV that would be rapidly degraded (FV is also called“labile factor” due to its low stability).

Example 5. Enrichment of the enzymatic factors II, V, VII, VIII, IX and X.

We used an automated instrument of our making to concentrate approximately 126 ml of plasma into approximately 20 ml of a coagulation complex concentrate, which consists of FII, FV, FVII, FVIII, FIX and FX. All factors are concentrated in their native zymogen/proenzyme form and do not undergo activation as part of the process.

We have previously defined ESC (Extrinsic System Concentrate) as a composition containing the factors II, V, VII, and X. These are the factors that define the extrinsic system of coagulation.

We now show herein that we are able to also extract factors VIII and IX with high yields. The definition of the extrinsic system is used for convenience, due to the ability to separately activate these factors in vitro as is reflected in the testing methods (PT for“extrinsic” coagulation factors vs. aPTT for“intrinsic” coagulation factors). Factors VIII and IX, however, can also be activated by the Tissue Factor pathway (the “extrinsic” pathway; Osterud, B., and Rapaport, S. I. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5260-5264). Therefore an ESC composition can also contain these factors. For convenience, we will refer to ESC containing also factors VIII and IX as CCC - Coagulation Complex Concentrate.

Materials and methods:

An anion exchange column (HiTrap Q Fast Flow) was used either in our automated FuseX system described herein, or in a standard FPFC instrument, together with Fresh Frozen Plasma and the herein described buffers.

Wash buffer: a buffer comprised of lOmM arginine, lOmM sodium citrate, and lOOmM NaCl pH 7.4

Elution buffer:“ 420mM NaCl“

Samples were loaded (126 ml ~ 25 column volumes) followed by wash with a wash buffer (15 ml ~ 3 column volumes) and an elution step (20 ml ~ 4 column volumes). In this example, we tested both the input plasma and the extract for each factor compared to an international standard. Since individual plasma units can differ in the levels of the individual factors, it is possible to have a concentration factor that is higher than the absolute concentration of factor, for example in the case where the starting levels of the said factor in the input plasma were lower than the international standard (or vice versa). Results are given in the format of % activity, both in the input plasma and the concentrated CCC, compared to the levels found in an international standard. For example, in the first run the concentration of factor II in the input plasma was 104.1% compared to the international standard (or 1.041 times as much), and the concentration of the same factor in the CCC was 517.2% (or 5.172 as much). That indicates that the CCC has approximately five times the activity of factor II per ml than the plasma that was used to make it, and (in this case) also over five times the activity of factor II than would be found in an international standard. In the same run, factor X levels in the input plasma were only 75% of the levels found in international plasma standard, and the CCC had approximately 400% (or 4-times as much) as the levels of the international standard per ml, but over five times as much concentration of active factor than the input plasma (402%/75%).

PROTOCOL: 25 column volumes of plasma were passaged on an anion exchange resin, followed by 2-3 column volumes of a low saline wash solution (<l50mM NaCl), and eluted with an intermediate NaCl concentration salt solution (350-600mM NaCl). The volumetric concentration was therefore approximately 6.3 fold (126/20).

We obtained the following data (Table 10) for the enzymatic factors II, VII, and X, and for the co-factor V.

Another experiment tested also the presence of the factor VIII as well as for the other factors (Table 11).

Therefore, all factors, both enzymatic factors and non-enzymatic co-factors, of the intrinsic tenase, extrinsic tenase, and prothrombin complex, are enriched over their initial concentration in plasma.

Demonstration of recovery of factor IX in CCC:

To prove that FIX is recovered at high concentrations in our standard extraction method we performed an extraction as described above and measured factor IX levels. Once again, the results show the factor IX activity compared to an international standard plasma..

The test is a aPTT-based assay (explained above). The recovery value is calculated as an average of the result from several dilutions that yield a similar recovery and are therefore on the linear part of the curve (<10% difference between measurements). In this case, three different measurements gave recovery yields of 79- 88% for FIX between different tested dilutions of ESC (Fig. 5).

In this protocol the volumetric reduction of the extract was approximately 12-13 fold (a volume of 126 ml of plasma was concentrated to 10 ml of extract). The results clearly show that Factor IX is recovered at very high levels (>80%), which is more than 10-fold (1000%) increase over the average factor IX plasma concentration.

Conclusion: The composition contains FIX at very high amounts, together with FII, FV, FVII, FVIII, and FX (tested in other experiments).

Example 6. CCC is beneficial in adhesion prophylaxis and fibrinogen reduction increases its specificity in a rat model.

The Coagulation Complex Concentrate (CCC) consists of FII, FV, FVII, FVIII, FIX and FX as obtained by the method in Example 1 or 6. In order to test the hypothesis that: a. a CCC would be beneficial in adhesion prophylaxis, and b. that Fibrinogen reduction would increase its specificity, we generated a CCC mix and applied it to rats who had undergone intensive abdominal surgery: midline (4 cm) incision followed by abrasion of the cecum for 30” with a coarse toothbrush to generate punctate bleeding, and performed a peritonectomy of a lxl cm square. The rats were allowed to heal for two weeks and sacrificed to analyze adhesion formation. Both adhesion area (Fig. 6A) and scores (area X grade; Fig. 6B) were measured. In one group, the CCC was supplemented with cryoprecipitate (20% v/v) in order to supplement Fibrinogen to the CCC (Figs. 6A-B).

As can be seen, while the test material was highly effective in reducing adhesions, the addition of cryoprecipitate - concentrated Fibrin, greatly reduced this effectiveness. Both the score and the area of adhesions were increased, indicating that the spread of Fibrin is correlated with increasing adhesions, and that a Fibrin-depleted CCC preparation is indeed the optimal solution to surgical adhesions.

Example 7. A stable CCC/ESC composition containing factor V.

While most coagulation factors (II, VII, and X) are relatively stable in their frozen stage, the same cannot be said of FV.

Still, using a single step-purification and concentration protocol, very high recoveries of FV (as high as >50% as in the next example) are able to be recovered. Furthermore, due to the minimal processing and purification

FV is relatively stable in plasma (Fresh Frozen Plasma = FFP is maintained routinely for as much as 2 years frozen without losing significant activities of factor V activity, as is known in the art). There is no commercial process available for producing stable FV. This is due to excessive purification, as stabilizing elements may be purified out.

In our system, however, FV is purified together with additional factors as part of an active ESC complex in a single step, as fast as 2 hrs. This composition, prepared in our system, is stable for at least 48 hours at cold (4 degrees Celsius) storage:

ESC (Extrinsic System Concentrate) is defined as a composition comprising the coagulation factors of the Extrinsic system, namely factors II, V, VII and X. CCC (Coagulation Complex Concentrate) refers to a wider definition of this system that includes factors VIII and IX (the“tenase” complex) as well, as these can also be activated by the Tissue Factor pathway.

Factors II, VII, IX, and X (also called the PPSB factors) are highly similar proteins sharing many traits. Factor V is the closest homologue to factor VIII.

While stable concentrates containing all other factors above are well known, factor V is notoriously known as a labile factor. In fact, no factor V containing product is available today (National Hemophilia Foundation), in no small part due to the difficulty in its stabilization. We therefore assessed the stability of the ESC/CCC based on the stability of the labile factor V.

Surprisingly, shock freezing of the composition at temperatures <-60C resulted in a massive decrease in their activity (at least 50%) (Fig. 7).

Freezing at -60°C for 30 minutes followed by storage at -30°C for 24 hours or 48 hours resulted in a decrease of FV recovery from about 58% to about 10-11%. At cold storage there is a minor decrease between 24 and 48 hours, whereas a much more dramatic decrease over time is seen at room temperature.

Liquid N2 freezing of the composition barely improved this data. Factor V activity in the ESC extract after liquid N2 freezing was reduced 51% (to 49% of its activity pre-freezing).

A viable option for the ESC is cold-storage transport chain. This is because blood hanks and establishments produce such labile products as platelets (In a closed system, current packs allow storage at 22°C ± 2°C with continual gentle agitation for up to 5 days; British Journal of Haematology, 2003, 122, 10-23, Guidelines for the Use of Platelet Transfusions).

The main reason for factor V degradation under non-frozen conditions is its activation to the highly labile active (Va) form. Therefore we used a small amount of Heparin sufficient to inhibit 1% or 5% of the activity of the coagulation factors (i.e., 0.01 or 0.05 heparin units/ml, respectively). This is to prevent the onset of the positive feedback loop of the coagulation pathway activation (leading to the complete conversion of the proenzymes to their active form), while not inhibiting the majority of the active composition. Heparin is an approved pharmaceutical and is therefore possible to use in a clinical setting.

Although a marked degradation of factor V is seen within 48 hours at cold (4C) storage, the addition of Heparin stabilizes approximately 75% of the activity within 48 hours, and 50% within 5 days. The level of factor V activity then stabilized at 25% for up to 30 days (longest timepoint tested). There was no advantage to the 5% (0.05 unit/ml) vs. 1% (0.01 unit/ml) heparin. Without heparin, within one week of the preparation the residual fibrinogen in the composition clotted and the solution was no longer liquid and testable (Fig. 8).

No data are available on the relative levels of factor V required for its full biological activity. Based on the similarity of factor V to factor VIII, its closest homologue, between 5-10% activity should be sufficient for full biological activity. Given a minimal yield of approximately 15% of factor V (lowest recovery level recorded in all of our experiments), this indicates that the material should still be fully biologically active after 5 days, and retain most of its biological activity up to 30 days, with the addition of a small amount of Heparin sufficient to inhibit just 1 % of the activity of the coagulation factors (0.01 unit of Heparin per 1 ml of input plasma used).

Another option to stabilize factor V is by improving the freezing process using cryoprotectants. These are pharmaceutically accepted excipients that have an effect on the freezing process and thereby on the composition. Surprisingly, in spite of the dramatic reduction of the factor V activity in ESC after freezing at low temperatures, we found that the addition of sucrose to a final concentration of approximately 2% (weight/volume) to the ESC composition and gentle freezing of the formulation at -30°C enabled full preservation of the factor V activity after as much as 4 weeks.

FV levels in ESC after freezing at -30°C with the addition of 2% sucrose (w/v) remain stable. A trendline shows no reduction in activity over the time tested. Without the addition of sucrose or with 3% sucrose, an initial decline of almost 20% is seen (Fig.

9).

The importance of the addition of the cryoprotectant at the correct concentration is evident by the fact that at the first time point at which FV activity levels were measured after freezing, there was a decline of almost 20% of activity in FV, both when no sucrose was present or when sucrose was at 3% (w/v) concentration. There was no further decline which proves that the effect is due to the freezing/thawing of the composition.

Thus, we have managed to create the world’s first stable FV containing concentrate. FV can be sufficiently stabilized to retain the majority of its biological activity without freezing which is a large advantage for some applications, and essentially completely stabilized for the long term by selecting the freezing method and cryoprotectant. Using 2% sucrose as a cryoprotectant and freezing at -30°C instead of at temperatures lower than -60°C (or liquid N₂) also protected against the large decline in activity of FV seen using other freezing methods.

Example 8. CCC is beneficial in adhesion prophylaxis and fibrinogen reduction increases its specificity in a pig model.

The Coagulation Complex Concentrate (CCC) comprises the intrinsic tenase, extrinsic tenase, and prothrombinase complex, i.e. FII, FV, FVII, FVIII, FIX and FX as obtained by the method in Example 1, 5, 6 or 7.

Female pigs underwent both abdominal and uterine surgery. CCC was prepared and applied directly in the peritoneal cavity on all regions operated. Pigs were allowed to recover for two weeks and then sacrificed for analysis.

Results: As can be seen in Figs. 10A-B, there was a dramatic and overall statistically significant reduction of both the score (severity) and extent (area) of the adhesions. Fig. 10A parses the score of the adhesions according to the region they were found in. In all regions there were less adhesions. In Fig. 10B all of the adhesion areas and scores were summed in the test and control pigs to yield a reduction of adhesions in both parameters that is larger than 80% and statistically significant (Student’s T-test).

In conclusion, CCC was efficient in reducing surgical adhesions in both the abdominal cavity and in gynecological procedures.

Claims

1. A sterile closed-system for producing a purified, enriched, therapeutic blood product, the system comprising a sterile chromatography system comprising:

(i) at least one separation module having fluid entrance and exit;

(ii) at least one sterile container comprising or designed to contain sterile wash solution fluidly connected to said fluid entrance of said separation module;

(iii) at least one sterile container comprising or designed to contain sterile elution solution fluidly connected to said fluid entrance of said separation module;

(iv) at least one sterile collecting container fluidly connected to said fluid exit of said separation module;

(v) a waste container and/or a waste exit fluidly connected to said fluid exit of said separation module;

(vi) a sterile connector suitable for sterilely connecting to a sterile container comprising plasma;

(vii) at least one stopcock; and

(viii) at least one one-way valve placed between at least some of the containers, at least one separation module, sterile connector and stopcock, ensuring directional process flow between the above containers, at least one separation module, sterile connector and stopcock;

2. The sterile closed system of claim 1, further comprising at least one pump designed to transfer the plasma, sterile wash solution, and sterile elution solution from their containers onto the at least one separation module without directly contacting the plasma, sterile wash solution, or sterile elution solution.

3. The sterile closed system of claim 1, further comprising a control module designed to control the operation of said at least one pump.

4. The sterile closed system of claim 1, further comprising a display unit.

5. The sterile closed system of claim 1, wherein each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising a resin selected from an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin.

6. The sterile closed system of claim 5, wherein each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising a resin selected from an anion exchange resin and a gel filtration/size exclusion resin.

7. The sterile closed system of claim 5 or 6, wherein the anion exchange resin is selected from DEAE, QAE, and Q; the cation exchange resin is selected from CM, S, SM and SP; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose.

8. The sterile closed system of claim 7, wherein the anion exchange resin is Q.

9. The sterile closed system of claim 1, wherein the sterile connector is suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RF- reactive thermoplastic material.

10. The sterile closed system of claim 1, wherein the at least one sterile container of

(ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump.

11. The sterile closed system of claim 10, wherein the at least one container designed to contain wash solution is one syringe operationally connected to one of the at least one syringe pumps, and the at least one container designed to contain elution solution is one syringe operationally connected to one of the at least one syringe pumps.

12. The sterile closed system of claim 1, wherein the at least one sterile collecting container is at least two sterile collecting containers fluidly connected to said fluid exit of said separation module via a manifold, wherein each sterile collecting container is connected to the manifold via a tube and a stopcock.

13. The sterile closed system of claim 1, wherein the stopcock connects one of the at least one separation module and the waste container and controls flow to either one of them.

14. The sterile closed system of claim 1, further comprising a power supply unit.

15. The sterile closed system of claim 1, wherein the sterile closed system further comprises at least one pump designed to transfer the plasma, sterile wash solution, and sterile elution solution from their containers onto the at least one separation module without directly contacting the plasma, sterile wash solution, or sterile elution solution; the sterile closed system further comprises a control module designed to control the operation of said at least one pump; the sterile closed system further comprises a display unit; each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin; the sterile connector is suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RF- reactive thermoplastic material; the at least one sterile container of (ii), (iii) or (iv) is a syringe; and the at least one pump is at least one syringe pump; the at least one sterile collecting container is at least two sterile collecting containers fluidly connected to said fluid exit of said separation module via a manifold, wherein each sterile collecting container is connected to the manifold via a tube and a stopcock; and the stopcock connects one of the at least one separation module and the waste container and controls flow to either one of them.

16. The sterile closed system of claim 15, wherein each one of the at least one separation modules is independently selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin; the at least one container designed to contain wash solution is one syringe operationally connected to one of the at least one syringe pumps, and the at least one container designed to contain elution solution is one syringe operationally connected to one of the at least one syringe pumps.

17. The sterile closed system of claim 16, wherein the anion exchange resin is selected from DEAE, QAE, and Q; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose.

18. The sterile closed system of claim 17, wherein the anion exchange resin is Q.

19. The sterile closed system of any one of claims 1 to 18, comprising a sterile connector suitable for sterilely connecting to the sterile container via sealed infusion tubing comprising RF -reactive thermoplastic material; a first syringe operationally connected to one of the at least one syringe pumps; the first syringe is connected via a one-way valve to a second syringe comprising washing buffer and operationally connected to one of the at least one syringe pumps; the second syringe is connected via a one-way valve to a third syringe comprising elution buffer and operationally connected to one of the at least one syringe pumps; the third syringe is connected via a one-way valve to a first separation module comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin and optionally to a fourth syringe comprising elution buffer and operationally connected to one of the at least one syringe pumps; the fourth syringe, if present, is connected via a one-way valve to the first separation module; and the first separation module is connected via the stopcock to the waste container and via the stopcock and a manifold to at least two collecting syringes, wherein each collecting syringe is connected to the manifold via a tube and a stopcock.

20. A method for producing a therapeutic dose of a purified, enriched, therapeutic blood product, the method comprising:

(a) sterilely connecting a first container comprising at least one unit of sterile blood plasma to the sterile chromatography system of any one of claims 1 to 19 using a sterile connection device;

(b) separating and enriching one or more therapeutic blood factors according to a predetermined program comprising loading the at least one unit of sterile blood plasma on one of the least one separation module by means of one of the at least one pump in combination with at least one one-way valve, thereby binding one or more therapeutic blood factors on the first separation module, washing the first separation module by transferring a first washing buffer from one of the least one sterile container comprising washing buffer onto the separation module by means of one of the at least one pump in combination with at least one one-way valve, and eluting a fraction from the separation module by transferring from one of the least one sterile container comprising elution buffer onto the separation module a first elution buffer, which optionally comprises an activating agent, by means of one of the at least one pump in combination with at least one one-way valve, wherein the first fraction comprises one or more therapeutic blood factors; and

(c) collecting the fraction comprising the one or more therapeutic blood factors in one of the at least one collecting containers and optionally freezing the fraction, thereby producing the purified, enriched, therapeutic blood product.

21. The method of claim 20, wherein the at least one unit of sterile, plasma, is (a) autologous plasma; (b) allogeneic plasma from a single donor that is pre-tested for pathogens; or (c) pooled allogeneic plasma that is pretested for pathogens and treated for viral reduction.

22. The method of claim 20, wherein the sterile connection device is a sterile tubing welder and the step of sterilely connecting the first container to a sterile chromatography system without compromising sterility of the unit comprises welding a tube comprising RF-reactive thermoplastic materials connected to first container to a tube comprising RF- reactive thermoplastic materials connected to the chromatography system by the means of the sterile tubing welder.

23. The method of claim 20, wherein each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin.

24. The method of claim 23, wherein the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin.

25. The method of claim 23 or 24, wherein the anion exchange resin is selected from DEAE, QAE, and Q; the cation exchange resin is selected from CM, S, SM, or SP; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose.

26. The method of claim 25, wherein the anion exchange resin is Q.

27. The method of claim 20, wherein the at least one sterile container of (ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump.

28. The method of claim 27, wherein the at least one container comprising wash solution is one syringe operationally connected to one syringe pump, and the at least one container comprising elution solution is one syringe operationally connected to one syringe pump.

29. The method of claim 20, wherein the wash solution comprises up to 200m M anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate; and a biocompatible buffer such as aliphatic acids.

30. The method of claim 20, wherein the elution buffer comprises between 200-2000 mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate.

31. The method of claim 30, wherein the elution buffer comprises between 380-500 mM NaCl or 25 mM CaCl₂ essentially without additional different chloride salt; and arginine and citrate.

32. The method of claim 20, wherein the at least one unit of sterile plasma is (a) autologous plasma; (b) allogeneic plasma from a single donor that is pre-tested for pathogens; or (c) pooled allogeneic plasma that is pretested for pathogens and treated for viral reduction; the sterile connection device is a sterile tubing welder and the step of connecting the first container to a sterile chromatography system without compromising sterility of the blood plasma or chromatography system comprises welding a tube comprising RF-reactive thermoplastic materials connected to the first container to a tube comprising RF-reactive thermoplastic materials connected to the chromatography system by the means of a sterile tubing welder; each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin; a cation exchange resin; a resin bound to an affinity ligand, such as an antibody, protein A, and protein G; and a gel filtration/size exclusion resin; the at least one sterile container of (ii), (iii) or (iv) is at least one syringe; and the at least one pump is at least one syringe pump; the wash solution comprises up to 200m M anions; and a biocompatible buffer such as aliphatic acids; and the elution buffer comprises between 200-2000 mM anions, such as chloride, citrate, phosphate, carbonate, sulfate, and sulfonate.

33. The method of claim 32, wherein each one of the at least one separation modules is selected from a column, filter, membrane or manifold comprising an anion exchange resin and a gel filtration/size exclusion resin; the at least one container comprising wash solution is one syringe operationally connected to one pump, and the at least one container comprising elution solution is one syringe operationally connected to one pump; and the elution buffer comprises between 380-500mM NaCl or 25 mM CaCE essentially without additional different chloride salt; and arginine and citrate.

34. The method of claim 33, wherein the anion exchange resin is selected from DEAE, QAE, and Q; and the filtration/size exclusion resin is selected from sephadex, agarose and sepharose.

35. The method of claim 34, wherein the anion exchange resin is Q.

36. The method of any one of claims 20 to 35, wherein the predetermined program comprises loading the blood plasma on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, washing the column by transferring a washing buffer comprising 100m M NaCl, lOmM arginine and lOmM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, eluting a fraction from the anion exchange column by transferring an elution buffer comprising 420mM NaCl, I OmM arginine and 10m M citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X, thereby producing the single therapeutic dose of a therapeutic blood product comprising non-stabilized, purified, enriched Factor II, Factor V, Factor VII, Factor VIII, Factor IX, and Factor X.

37. The method of any one of claims 20 to 35, wherein the predetermined program comprises loading the blood plasma on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, washing the column by transferring a washing buffer comprising 100m M NaCl, 10m M arginine and 10m M citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, eluting a fraction from the anion exchange column by transferring an elution buffer comprising 300mM NaCl, I OmM arginine and I OmM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in the first of the at least one collecting container, wherein the fraction comprises purified and enriched Factor VII thereby producing the single therapeutic dose of a therapeutic blood product comprising a purified, enriched Factor VII.

38. The method of claim 37, wherein the recovery efficiency is at least 15%, preferably at least 40%, the concentration factor is at least 200%, preferably at least 500%, and the relative purity is at least 80%.

39. The method of any one of claims 20 to 35, wherein the predetermined program comprises loading the blood plasma on a column comprising the anion exchange resin Q by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, washing the column by transferring a washing buffer comprising 75mM NaCl, I OrnM arginine and I OrnM citrate, at physiological pH, such as 7.4, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, activating and eluting a bound fraction on the anion exchange column by transferring a buffer comprising at least l5mM CaCl₂, preferably 25mM CaCF, and 10m M arginine and 10m M citrate, at physiological pH, such as 7.4, essentially without additional different chloride salt, by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises purified and enriched Factor Vila thereby producing the single therapeutic dose of a therapeutic blood product comprising a purified, enriched Factor Vila.

40. The method of claim 39, comprising eluting a second fraction from the column with an elution buffer comprising about 300mM NaCl and I OrnM arginine and 10m M citrate, at physiological pH, such as 7.4 by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises purified and enriched Factor X, thereby producing the single therapeutic dose of a therapeutic blood product comprising a purified, enriched Factor X.

41. The method of claim 39, comprising eluting a second fraction from the column with an elution buffer comprising about 420mM NaCl and 10m M arginine and lOmM citrate, at physiological pH, such as 7.4 by means of a syringe operationally connected to one of the at least one syringe pump in combination with at least one one-way valve, and collecting the fraction in one of the at least one collecting container, wherein the fraction comprises purified and enriched Factor X and Factor II, thereby producing the single therapeutic dose of a therapeutic blood product comprising a purified, enriched Factor X and Factor II.

42. The method of any one of claims 20 to 41, wherein the blood product is a labile and non-stabilized blood product.

43. The method of any one of claims 20 to 41, further comprising stabilizing the blood product by adding a cryoprotectant, such as sucrose, mannitol, trehalose, polyethyleneglycol (PEG), and glycine; or an anticoagulant, such as heparin or a derivative thereof and enoxaparin sodium, and storing it, thereby producing a stabilized blood product.

44. The method of claim 43, wherein the cryoprotectant is sucrose at a final concentration of about 2% (w/v) and the method further comprising freezing the stabilized blood product by exposing it to a temperature of -30°C and subsequently storing it at about -30°C.

45. The method of claim 44, wherein the blood product, after storing up to about four weeks or longer, comprises Factor V having a level of activity that is essentially equal to freshly produced Factor V.

46. The method of claim 43, wherein the anticoagulant is heparin at a final concentration of about 0.01 unit/ml; and the method further comprising storing the stabilized blood product at about 2-8°C.

47. The method of claim 46, wherein the blood product, after storing up to about four weeks or longer, comprises Factor V having a level of activity that is about 25% of that of freshly produced Factor V.

48. The method of any one of claims 20 to 47, wherein the blood product is essentially devoid of hemoagglutinins.