WO1999020288A1

WO1999020288A1 - Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma

Info

Publication number: WO1999020288A1
Application number: PCT/US1998/021626
Authority: WO
Inventors: Lou Blasetti
Original assignee: Harvest Technologies Corporation
Priority date: 1997-10-17
Filing date: 1998-10-16
Publication date: 1999-04-29
Also published as: JP2001520198A; CN1113656C; EP1023078A1; EP1023078A4; CA2306629A1; CN1276725A

Abstract

Increased fibrinogen yields are obtained by adding a precipitating agent to plasma having a high platelet concentration, such as platelet rich plasma. The precipitating agent may be any of several known agents, including polyethylene glycol and ammonium sulfate. The platelet rich plasma is obtained in the preferred embodiment by subjecting plasma to 'soft spin' centrifugation of about 580G. An automatic, multiple decanting and multiple-speed centrifuge is preferably used to separate anti-coagulated whole blood into the platelet rich plasma component and red blood cells. The proteins, preferably fibrinogen, FXIII, and FVIII, in the platelet rich component are precipitated, and the proteins and platelets are then concentrated by further centrifugation.

Description

PRECIPITATION OF GROWTH-FACTOR-ENRICHED FIBRINOGEN

CONCENTRATE FROM PLATELET RICH PLASMA

TECHNICAL FIELD

This application relates to improved processes for recovery and concentration

of blood components. In particular, the invention relates to the production of growth-

factor-enriched fibrinogen concentrate from platelet-rich plasma.

BACKGROUND

The need exists for means quickly to concentrate and recover certain blood

proteins from whole blood, which also contains platelets and certain growth factors,

in a closed-process system for use by physicians to assist in closing wounds, to

achieve faster haemostasis, to seal air and fluid leakage, and to aid in faster healing

and for drug and biologic delivery.

Those skilled in the art know that when platelet-rich plasma is harvested from

a surgical patient intraoperatively and is combined with thrombin, usually in a seven-

to-one ratio, and deposited on a wound site, a platelet gel is formed within seconds

of application. The gel achieves faster haemostasis than do other conventional

haemostatic agents. The gel also seals air and fluid leakage due to its viscous

properties, and results in faster healing resulting from the presence of platelet

derived growth factors (PDGF). Such a gel contains only native levels of fibrinogen,

FXIII, FVIII, and PDGF. Thus, the adhesive, tensile and shear strength of the clot formed by this gel is generally less than is desirable. Further, failure of haemostasis

or sealing can occur because of these low levels of desirable proteins, resulting in a

failure to achieve the desired outcome.

Harvesting platelet rich plasma from a patient in the intra-operative setting

requires a "blood processor," one of which is sold under the trademark "Cell Saver,"

but other devices manufactured by various companies are known. The Cell Saver

device requires a highly-skilled, sometimes certified, operator to set-up and operate

the device. Operation (which can take 30 to 60 minutes) requires large-bore venous

or arterial access and processing of up to several liters of biood to obtain and

sequester sufficient platelets and plasma volume. The patient's haemodynamic and

cardiac status must be stable to allow processing of such large volumes.

An automated system for obtaining autologous fibrinogen has been described

in United States Patent 5,707,331 (Wells et al.), the disclosure of which is

incorporated herein by reference. According to that system, a relatively small

volume (e.g., 50ml) of whole blood is placed in a first chamber of a two-chamber

disposable container. A fibrinogen-precipitating agent is placed in the second

chamber. The container is then placed in a centrifuge, and the whole blood is

centrifuged to separate the plasma to produce platelet-poor plasma. The platelet-

poor plasma, thus obtained is then decanted into the second chamber where it is

mixed with the precipitating agent (e.g., PEG or ammonium sulfate). The plasma

and precipitating agent are then centrifuged to obtain a pellet of fibrinogen for

combination with thrombin to make a fibrin sealant. DESCRIPTION OF THE PREFERRED EMBODIMENT

An important factor for processes that recover fibrinogen, such as the one

described in the mentioned United States Patent 5,707,331 , is the percentage of the

fibrinogen in the whole blood that is recovered in the pellet. Applicant has

discovered that this factor, the "fibrinogen yield," is unexpectedly greater when the

plasma from which fibrinogen is precipitated contains increased levels of platelets.

Thus, according to the process of the invention, a known fibrinogen precipitating

agent is added to platelet-rich plasma to obtain increased yields of fibrinogen.

The fibrinogen yield obtained with prior art methods is generally about 50%,

whereas the fibrinogen yield obtained in accordance with the methods of the

invention is about 72%, which represents a 44% increase in recovered fibrinogen.

In the preferred embodiments, the platelet-rich plasma from which fibrinogen

is precipitated contains at least 50K platelets per mm³ and preferably about

200K/mm³.

The disclosed invention produces FVIII and concentrated (up to 10+ fold

increase) proteins, preferably fibrinogen, FXIII, and recovered platelets (and

resultant increase in human growth factors) from relatively small aliquots (20cc-

150cc) of anti-coagulated whole blood in a short time (approx. 20 minutes). The

increased coagulation protein concentration of the disclosed invention over the

current Cell Saver methods results in a clinically more effective (greater tensile and

shear strength) clot. A clinically effective dose is produced from a smaller volume

(20cc-150 cc) of the patient's blood obtained by simple phlebotomy known in the art

versus the Cell Saver method (several liters). The preferred method utilizes the dedicated centrifuge and disposable

container described in United States Patent 5,707,331 to process anti-coagulated

whole blood drawn from a patient (or directed blood donor). In accordance with the

invention, the process described there is modified to provide platelet-rich plasma by

appropriate control of the centrifuge speed and the length of time the blood is

subjected to centrifugation.

Anticoagulated blood retrieved from a mammal by simple phlebotomy

techniques is dispensed into a first chamber of a 2-chamber disposable, and an

appropriate volume of a precipitating agent, for example PEG or saturated

ammonium sulfate, is placed in the second chamber. The ammonium sulfate can be

25% to 100% ammonium sulfate, and is preferably about 95% ammonium sulfate.

The disposable is loaded into the dedicated centrifuge as described in United States

Patent 5,707,331 , and the process in that patent initiated. The centrifuge is

programmed to effect the following steps automatically:

1. Red cells are separated from whole blood in the centrifuge at a spin rate that

produces platelet-rich plasma (PRP). The spin rate is known as a "soft spin"

and preferably one that produces about 580G. The centrifuge is then

stopped, and the PRP is decanted from the first chamber to the second,

where it is mixed with the precipitating agent. This soft spin has been found

to produce plasma having a platelet concentration of from about 50K/mm³ to

about 450K/mm³.

2. After mixing is complete, the centrifuge re-starts and the precipitated proteins,

along with the platelets, are concentrated by a "hard spin," preferably one that

produces about 3500G. 3. Following step 2 above, the platelet-poor and fibrinogen-poor plasma and

residual precipitating agent are decanted from the second chamber back to

the first, leaving a relatively-dry, growth-factor-enriched protein/platelet pellet.

The use of a precipitating agent, such as PEG or ammonium sulfate, with

PRP has been found to provide greater protein (preferably fibrinogen)

recovery than obtained with techniques using a precipitating agent with

platelet poor plasma (PPP).

4. A suitable diluent volume, preferably a citrate buffer, is added to re-dissolve

and recover the protein/platelet pellet to allow transport by, for example,

syringe.

5. When the recovered, concentrated protein, containing increased levels of

human growth factors, is combined with thrombin and deposited on a wound

site, a platelet gel is formed within seconds of application. The gel achieves

faster haemostasis than when other conventional haemostatic agents are

used. It can also seal air and fluid leakage due to its viscous properties, and

results in faster healing from the presence of enriched platelet derived growth

factors (PDGF). The gel's properties include FVIII and increased levels of

fibrinogen, FXIII, and greater than native levels of human growth factors.

These increased levels result in a clot with more desirable adhesive, tensile

and shear strength. Because of these^' higher levels of desirable proteins, the

risk of premature failure of the clot is reduced and the likelihood of achieving

the desired outcome is increased. Example 1.

Fifty milliliters of whole blood were placed in the first chamber of a container

for use in an automated centrifuge, and 15 milliliters of 30% polyethylene glycol

(MW1000) were placed in the second chamber. The container was then subjected

to a soft spin of about 580G for three minutes. The platelet-rich plasma thus

obtained (23-25ml) was then decanted to the second chamber and mixed with the

PEG. The container was then subjected to hard centrifugation and the supernatant

was decanting back to the first chamber. The result was a fibrinogen pellet

representing a fibrinogen yield of approximately 70%, a four-to-ten fold increase in

TGF-B-1 and a thirty-fold increase in PDGF-AB.

Example 2.

for use in an automated centrifuge, and 7ml of saturated ammonium sulfate was

placed in the second chamber. The container was then subjected to a soft spin of

about 580G for three minutes and 23-25 milliliters of platelet-rich plasma were

decanted to the second chamber. After mixing with the platelet-rich plasma with the

ammonium sulfate, the container was subjected to a hard spin to obtain a fibrinogen

pellet, and the supernatant decanted to the first chamber. The fibrinogen yield of the

pellet was about 72% a four-to-ten fold increase in TGF-B-1 and a thirty-fold

increase in PDGF-AB.

Modifications within the scope of the appended claims will be apparent to

those of skill in the art.

Claims

I Claim:

1. A process for isolating growth factor enriched fibrinogen concentrate

comprising the steps of:

obtaining platelet rich plasma,

adding a fibrinogen-precipitating agent to said platelet rich plasma; and

recovering growth factor enriched fibrinogen concentrate from said platelet

rich plasma.

2. A process according to claim 1 wherein said precipitating agent is

polyethylene glycol.

3. A process according to claim 1 wherein said precipitating agent is ammonium

sulfate.

4. A process according to claim 1 wherein said step of obtaining platelet rich

plasma comprises the step of subjecting plasma to centrifugation.

5. A process according to claim 4 wherein said step of centrifugation comprises

the step of subjecting said plasma to a force of about 580G for about three minutes.

6. A process according to claim 1 wherein said platelet rich plasma comprises

plasma having 50K to 450K platelets/mm³.

7. A process according to claim 1 wherein said step of recovering fibrinogen

comprises the step of subjecting said platelet rich plasma and said precipitating

agent to centrifugation.

8. A process according to claim 1 wherein the step of obtaining platelet rich

plasma comprises the step of subjecting about 50ml of anticoagulated whole blood to centrifugation and decanting 23-25ml of said platelet rich plasma, and said step of

adding a precipitating agent comprises adding about 15ml of 30% polyethylene

glycol (MW1000) to said platelet rich plasma.

9. A process according to claim 1 wherein the step of obtaining platelet rich

plasma comprises the step of subjecting about 50ml of anticoagulated whole blood

to centrifugation and decanting 23-25ml of said platelet rich plasma, and said step of

adding a precipitating agent comprises adding about 7ml of saturated ammonium

sulfate.

10. A process according to claim 1 wherein said step of recovering fibrinogen

further comprises the step of adding a buffer to said fibrinogen.

11. A product made by the process of any one of claims 1 -10.