Note: Descriptions are shown in the official language in which they were submitted.
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CARBOXYLIC ACID SUCH AS CITRIC ACID FOR DESINFECTING OR ENHACING THE
PRODUCTION OF
BLOOD PRODUCTS SUCH AS PLASMA, CRYOPRECIPITATE OR/AND PLATELET
Related Applications
The present application is a continuation in part of US Patent
Application Serial No. 09/694,178 filed October 23, 2000, and US Patent
Application Serial No. 09/778,681 filed on 7 February 2001 and US Patent
Application Serial No. 60/278,496 filed 23 march 2001 and claims priority
from the later two applications. All these applications are incorporated by
reference herein.
Background of the Invention
Area of the Art
The present invention relates to an improved method for producing
increased amounts of safe coagulation factor concentrates from blood
plasma.
The invention is also directed to enhancing the yield and purity of
blood components and inhibiting the activation or denaturation of certain
blood components, blood cells and plasma proteins, and to the removal of
activated and denatured components, 'thereby improving the safety and
efficacy of end products.
Description of the Prior Art
There are a number of medical indications for administration of
"clotting" or "coagulation" factors from human blood. These factors are
proteins that cause the clotting of blood to staunch bleeding from wounds,
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etc. Individuals with any of a series of genetic abnormalities affecting the
proteins responsible for blood coagulation are afflicted with a disease
(hemophilia) in which the blood fails to clot normally, subjecting the
individual to the danger of uncontrolled bleeding. For many years, this
condition has been treated by administering concentrates of the missing or
defective proteins. Many clotting factors are synthesized in the liver so that
victims of liver disease are also in need of augmentation of their clotting
factors. Additionally, there are other important medical uses for clotting-
related factors including the use of fibrin to produce "fibrin sealant" or
"fibrin glue".
While some of the clotting factors are currently produced through
biotechnology, at this time there is still no cost effective method of
artificially manufacturing all of these proteins or these proteins in
sufficient
quantities. Further, the "artificially produced" factors made by recombinant
and related technologies tend to be more expensive. Many of the "minor"
factors are not yet (and may never be) available from biotechnology sources
and so must be purified from donated human blood. This is especially true in
Third World countries where the biotechnology products are generally not
available or affordable. Therefore, much of the supply of anti-hemophilia
factor (AHF, also known as Factor VIII), and other blood clotting factors are
prepared from pooled human plasma. A hemophiliac requires treatment for a
whole lifetime. Victims of liver disease and other users of clotting factors
may also require prolonged treatment. Therefore, these patients are exposed
to blood products produced from the blood of a large number of donors.
The presence of AIDS (Acquired Immuno Deficiency Syndrome) virus
or HIV in the blood supply means that hemophiliacs and other users of
clotting factors have become infected with this terrible disease. Although
tests to screen out AI°DS-tainted blood have been improved, some
infected
blood does slip by. Even if the AIDS problem is solved, the danger of other
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blood-borne diseases, such as the various types of hepatitis and other, as
yet unknown, infectious agents, makes it desirable to reduce or eliminate
virus and other disease organisms from plasma used to prepare clotting
factors. One way of achieving this goal is to replace pooled plasma products
with products from a single donor since with pooled products "one bad
apple spoils the entire barrel". However, even with the use of clotting
factors derived from a single donor, there is still danger. Even though tests
may show the donor is free of known disease, the donor may be incubating
a disease that will later show on the tests, or the donor may harbor a yet
unknown disease or a yet unknown strain of a known disease. These
dangers have been lessened by use of plasma pre-treatments that inactivate
disease organisms. Unfortunately, the best commonly used treatments
either do not inactivate all types of disease organisms or damage the labile
clotting factors during the process of inactivating disease organisms.
The basic methods for preparing clotting factor concentrates from
blood have not changed greatly over the last few decades. Generally, a
concentrate of clotting factors is derived from pooled plasma by a
cryoprecipitation step. Various additives such as efihanol or polyethylene
glycol are usually added to enhance the efficiency of the cryoprecipitation
step. Following cryoprecipitation, the partially purified factors may be
further purified by additional precipitation steps or by chromatographic
methods, and most recently by methods using monoclonal antibodies. For
additional information on the basic techniques of clotting factor purification
and the History of the development of these methods, the reader is directed
to U.S. Pat. Nos. 3,560,475, 3,631,018, 3,682,881, 4,069,216, and
4,305,871 and 5,770,704 by the present inventor, the contents of which
are incorporated herein by reference, and the references cited therein.
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Summary of the Invention
It is an object of the present invention to enhance the yield and purity
of cryoprecipitate;
It is a further object of the present invention to inactivate and/or
enhance the inactivation of disease organisms within plasma at the same
time that cryoprecipitate production is enhanced and the cryoprecipitate is
further purified.
It is a further object of the present invention to inhibit the activation
or denaturation of blood components, including blood cells and plasma
proteins, and/or to remove these activated or denatured components,
thereby improving the safety and efficacy of the end product.
It is an additional object of the present invention to provide an
improved method for blood fractionation.
Derivatives of simple carboxylic acids, particularly trisodium citrate
and other citric acid salts (hereinafter "citrate") are shown to be
unexpectedly effective agents for enhancing the production of blood clotting
factors. It is believed that other small carboxylic acids, isocitric acid in
particular, may show similar properties. However, to date most of the tests
have been made with citric acid and its salts. Addition of citrate to plasma,
especially at concentrations between 2 and 10 % by weight, does not
appreciably denature Habile proteins. However, in this concentration range
citrate is effective in inactivating or inhibiting a variety of pathogenic
microorganisms. Further, the added citrate potentiates or enhances the
killing of microorganisms by heat treatment. That is, heating of the material
to relatively low temperatures (i.e., above 45°C) which do not denature
proteins enhances the killing of microorganisms in the presence of citrate.
Most significantly, added citrate causes a dramatic increase in the weight of
cryoprecipitate that can be produced from plasma by the usual procedures.
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The majority of significant clotting factors are greatly concentrated in the
resulfiing cryoprecipitate. The supernatant contains little if any of these
clotting factors. It is apparent that increasing the amount of citrate in
blood
bags so that the final concentration will be at least 2% by weight results in
5 plasma that can be used to produce improved platelet concentrates and
enriched cryoprecipitate. The added citrate can ,help eliminate or suppress
contaminating microorganisms and can itself be removed later by ion
exchange or similar methods well known in the art.
Another aspect of the invention is the use of citrate to enhance the
yield and purity of cryoprecipitate. Furthermore, added citrate can inhibit
the
activation or denaturation of blood components including blood cells and
plasma proteins and/or facilitate the removal of the activated or denatured
components and improves the safety and efficacy of end products.
According to the invention there is provided a method for reducing
transfusion-associated disease and adverse effects in plasma and for
enhancing the purity and safety of multiple derivative components of blood
including blood cells and plasma. In this method, there is the step of adding
at least about 2% by weight of carboxylic acid salt or equivalent weight of
carboxylic acid to the blood or plasma.
Moreover, the invention is directed to enhancing the production of
other derivative blood components including blood cells and plasma
proteins.
The invention includes reducing transfusion-associated disease and
adverse effects in plasma and for enhancing the purity and safety of
multiple derivative components of blood including blood Cells and plasma
from plasma. The derivatives comprises at least one product from the group
of Enriched Cryoprecipitate, Cryo-Depleted Plasma, Fibrinogen, Fibrin Glue
or Sealant, vWF (von Willdebrand's factor), Fibronectin, Factor VIII,
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Prothrombin Complex, a Serpine, Albumin, and a Globulin from plasma. At
least 2% by weight of a salt of citric acid (or equivalent weight of citric
acid) is added to the plasma. The plasma may be collected into a blood bag
containing the carboxylic acid or the carboxylic acid salt. This blood bag can
be different from a bag or container used to collect whole blood.
Alternatively or additionally, an amount of additional carboxylic acid or the
salt thereof may be added directly to the bag used to collect the whole
blood.
In a further preferred form of the invention, citrate is used
appropriately in the collection of blood, in the processing and transfer of
blood in a separation of discrete blood components. Citrate is used in
increased, namely, additional quantities over the level traditionally employed
for anticoagulation in one or other collection or processing bag.
Brief Description of the Figures
FIGURE 1 is a graphic representation of the improvement in
cryoprecipitate yield resulting from the present invention.
FIGURE 2 is a representation of the blood and plasma fractionation
scheme using the invention.
Detailed Description of the Invention
The following description is provided to enable any person skilled in
the art to-.make and use the invention and sets forth the best modes
contemplated by the inventor of carrying out his invention. Various
modifications, however, will remain readily apparent to those skilled in the
art, since the general principles of the present invention have been defined
herein specifically to provide enhanced production of plasma proteins along
with inactivation of blood borne disease organisms.
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The traditional method for producing clotting factors, as well as many
of the presently used methods, operate because many plasma proteins
responsible for clotting precipitate (i.e., form cryoprecipitate) from
solution
at low temperatures when they are sufficiently concentrated. When a
protein solution is frozen, ice crystals form and protein molecules, which are
excluded from the crystals become increasingly concentrated. Cooling or
freezing the water also lowers the chemical activity of the water. Depending
on the particular proteins, the proteins may actually fall out of solution,
i.e.,
form a precipitate, if the protein more readily interacts with itself or with
other proteins than with water. When the chemical activity of water is
lowered such precipitation is favored. This process may denature the
proteins (make them irreversibly insoluble), so it is usual to freeze protein
solutions rapidly and to a low temperature (i.e., -20°C. or lower) to
minimize the formation of ice crystals and to prevent the growth of those
crystals that do form. This is done to limit protein denaturation on ice
crystal surfaces. However, even when freezing is carried out with great
care, ice crystals may cause "activation" of the prothrombin complex,
resulting in spontaneous clot formation after the plasma is thawed.
The first step in the typical procedure for producing plasma
cryoprecipitate is to centrifuge whole blood to separate the plasma from the
red blood cells. This procedure is well known in the art and is often
accomplished in special centrifuges that hold individual blood bags so that
the plasmalred cell separation occurs without even opening the blood bag.
Following the centrifugation, it is common practice to express the
supernatant plasma into a "satellite" blood bag for further processing. Once
the plasma is separated from red and white blood cells, the typical
procedure is to rapidly freeze the plasma and to then slowly thaw the frozen
plasma at about 4°C., during which thawing the clotting factors and
other
proteins form a cryoprecipitate which can be readily harvested by filtration
or centrifugation. This cryoprecipitate is not rendered irreversibly insoluble
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and can be readily redissolved in a low ionic strength buffer, or even water,
as is well known in the art.
Cryoprecipitation is generally believed to result when the removal of
water from the immediate vicinity of the protein molecules causes the
protein molecules to preferentially associate with each other rather than
with water. This "removal" of water may represent changes in the solubility
of the proteins with changes in temperature (i.e., water becomes less
effective at dissolving the proteins). The process may also be accomplished
or enhanced by using additives which "tie up" the water and cause it to
interact with the proteins to a lesser degree. These additive substances can
be any of a number of hydrophilic materials such as ethanol, polyethylene
glycol, heparin, Pluronic RTM polyol polymers and various "salts" such as
ammonium sulfate or ammonium acetate. The "salting out" of proteins from
solution is a classical biochemical procedure. These and other materials used
to increase the yield of cryoprecipitate generally operate to decrease the
effective activity of water in the mixture. That is, the water molecules
preferentially interact with the added hydrophilic material instead of with
the
proteins. This permits the proteins to interact with each other and,
therefore, precipitate from solution. Similarly, lowering the temperature also
decreases the activity of water, allowing protein-protein interactions to
predominate.
The hydrophilic additives just mentioned have the advantage of being
relatively inexpensive and easy to use. However, their use usually
necessitates additional washing steps to ensure that the additives are not
carried over into fihe final product. Some additives may also damage or
denature the labile clotting factors one is seeking to purify. The present
inventor has discovered that one of the agents frequently used as an
anticoagulant in blood fractionation unexpectedly serves to enhance
cryoprecipitate formation. Citrate (trisodium citrate or similar salts as well
as
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derivatives of other low molecular weight carboxylic acids such as isocitric
acid) has unusually favorable properties when used in blood fractionation
procedures at levels significantly higher than those traditionally used as an
anticoagulant. Citrate is a fairly effective chelator of calcium ions. By
effectively lowering the calcium ion level, citrate inhibits a considerable
variety of blood clotting pathways which depend on the presence of calcium
ions. However, citrate has not been employed as an agent to enhance the
preparation of cryoprecipitate proteins from plasma and other blood
fractions.
The following table shows the enhanced production of cryoprecipitate
caused by increasing the level of trisodium citrate in plasma. As the citrate
is increased, the weight of recovered cryoprecipitate is increase. When the
cryoprecipitate is redissolved in a fixed quantity of buffer or water, fihe
increasing amount of cryoprecipitate yields increasing amounts of Factor VIII
and fibrinogen as compared to the original plasma. The precise reason for
this increase in yield is not known. However, it seems reasonable to
speculate that one action of citrate may be to inhibit the activation of
clotting factors. Since many of these factors act as proteases when
activated, activation naturally digests clotting proteins thus reducing the
yield of these proteins. However, lack of inactivation does not seem
sufficient to account for the entire increase in cryoprecipitate yield.
Treatment Cryoprecipitate Factor VIII Fibrinogen
control 0.1 g 120% 46 mg/dl
2% citrate ~ 0.3 g 180% 53 mg/dl
5% citrate 0.9 g 247% 105 mg/dl
10% citrate 3.0 g 622% 152 mg/dl
These data are graphically represented in Fig. 1. These results
indicate that as the citrate concentration is increased the amount of
recovered clotting factors increases linearly. However, at the highest
concentration of citrate it would appear that there might be an increase in
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the precipitation of other proteins. It may be possible to adjust the citrate
concentration to favor the precipitation of different proteins. Tests have
shown that besides more than 95% of the Factor VIII and Fibrinogen,
virtually all of the Fibronectin and the von Wiildebrand's factor become
5 concentrated in the citrate-enhanced cryoprecipitate. Additional experiments
have been undertaken to determine if metalloproteins or other factors are
preferentially concentrated in the citrate cryoprecipitate. Initial results do
not
show any other proteins as strongly concentrated as those already
mentioned. However, there is some indication that ceruioplasmin and total
10 T3 are somewhat concentrated in the cryoprecipitate.
On the surface, one might not expect citrate to be more effective
than any hydrophilic salt. In terms of salting proteins out of solution, one
would expect various agents to operate based on their colligative properties.
That is, one might expect equimolar concentrations of various agents to
behave similarly. This does not appear to be the case with citrate and
cryoprecipitate formation.
The following quantities of either salt (NaCI) or citrate (trisodium
citrate) were added to 40 ml aliquots of fresh human plasma. After
thorough mixing the samples were frozen overnight at -70°C and then
completely thawed at 4°C. The samples were then centrifuged at 4000
RPM for 20 min to harvest the cryoprecipitate. An attempt was made to
match the effective sodium concentration between the sodium chloride and
sodium citrate on the basis that each molecule of trisodium citrate would
provide three sodium ions whereas each molecule of sodium chloride would
provide only a single sodium ion. This attempt at compensation was
inaccurate because the matching should be done on a molar rather than a
percent basis. However, this failed experiment points out the incredible
superiority of sodium citrate over sodium chloride for producing
cryoprecipitate. In the following table the citrate (trisodium citrate) or
salt
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(sodium chloride) are shown, first as weight percentages and then as
molarities, The third column shows the effective osmotic effect of the
solutions, which is two times higher on a molar basis for citrate than for
sodium chloride. This is because each molecule of sodium chloride releases
only two particles (one sodium ion and one chloride ion) whereas each
molecule of trisodium citrate releases four particles (three sodium ions and
one citrate ion). Because the molecular weight of trisodium citrate is almost
5 times greater than that of salt to get equal osmotic effects one must use
about 2.5X (on a weight basis) as much trisodium citrate as sodium
chloride. That is, for an accurate matching more citrate rather than more
salt should have been used.
Weight % Molarity Osmotic Effect Cryoprecipita~te
Control -- -- 0.18g
2% citrate 0.07 0.28 0.52g
5% citrate 0.17 0.68 1.3g
10% citrate 0.34 1.36 3.6g
6% sodium chloride 1.02 2.04 0.12g
15% sodium chloride2.56 5.13 0.14g
30% sodium chloride5.13 10.26 0.15g
These results show that the effect of citrate on cryoprecipitate
production is not strongly related to the coiiigative or osmotic properties of
the citrate. Sodium chloride seems not to enhance cryoprecipitate
formation. Only at osmotic levels that are greatly above those of the
maximal citrate concentration, cryoprecipitate formation begins to approach
that of the control plasma. Further, the resulting cryoprecipitate does not
appear as pure (that is, larger amounts of other non-clotting proteins are
included).
Following the experiment, the supernatants and the original plasma
(control) were sent to a clinical chemistry laboratory to determine the
presence of various blood proteins including clotting factors. These results
are shown in the following table.
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Weight % Fibrinogen (mg/dl) Factor V111 (%) Albumin (gldl)
Control 287 24 3.2
2% citrate 216 6 3.1
5% citrate 1 14 < 3 3.2g
10% citrate < 40 < 3 3.2g
6% NaCI 298 30 3.1 g
15% NaCI 229 24 3.0g
30% NaCI 97 9 2.9g
As the amount of citrate is increased the levels of fibrinogen and Factor VIII
in the supernatant decrease dramatically. At the same time, the level of
albumin (the major plasma protein) is essentially unaffected. In other words,
most of the clotting factors precipitate and are found in the cryoprecipitate,
but little or no albumin precipitates. In the case of sodium chloride,
equimolar concentrations are much less effective at precipitating the clotting
factors. One has to go up to 30% sodium chloride to see a significant
precipitation of the clotting factors. However, at this level the albumin also
begins to precipitate. Citrate is far more effective at selectively
precipitating
the clotting factors.
Further insight into the citrate effect is gleaned by analyzing the
distribution of citrate in a typical cryoprecipitate experiment. For this
experiment, one unit (about 200 ml) of plasma was brought to 10% wt/vol.
trisodium citrate. In ali experiments pH measurements showed that natural
buffering of the plasma prevented significant changes in pH. This citrate-
treated plasma was frozen and cryoprecipitate was collected in the usual
manner. As an aside, in producing citrate cryoprecipitate it is preferred to
add the citrate prior to freezing, but good results are achieved by adding the
citrate during the thawing process.
The volume of cryoprecipitate formed from the unit of plasma was
approximately 20 ml-that is, 10% of the total volume. Surprisingly, an
analysis of the cryoprecipitate and the supernatant plasma showed that
about 12 g (60%) of the citrate was concentrated in the cryoprecipitate
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with only 30% being left in the supernatant, This indicates that there is a
strong interaction between the cryoprecipitate proteins and the citrate.
Further, while normal cryoprecipitate can be redissolved in room
temperature water or buffer, citrate cryoprecipitate is almost insoluble in
room temperature water. It is soluble, however, in room temperature saline
buffer and most soluble when the buffer contains citrate. One way of
explaining these phenomena is to assume that the multiple negative charges
on the citrate molecule are interacting with positive charges on the
cryoprecipitate proteins to cross-link them. Added citrate "satisfies" these
positive charges so that cross-linking is abolished. Because of the
concentration of clotting proteins into the cryoprecipitate, it is tempting to
theorize that all of the clotting proteins share some sort of positive charge
motif that interacts with the citrate molecules.
In summary, compared to "normal" cryoprecipitate citrate
cryoprecipitate contains essentially all of the Fibrinogen, Fibronectin,
Factor
VIII and von Willdebrand's factor found in a treated aliquot of plasma. The
citrate cryoprecipitate may also contain other minor factors (like Factor
XIII)
not yet assayed in these experiments. What may be significant is what the
citrate cryoprecipitate does not contain. It has significantly less of
albumin,
globulins and other minor proteins than "normal" cryoprecipitate.
Experiments are going on to characterize these differences.
Although citrate appears to influence strongly the precipitation of the
clotting factors, it does not appear to denature these proteins. Citrate at 2%
by weight was added to an aliquot of plasma that was stored at room
temperature for six days. Clotting factors and platelets were. counted at the
beginning and the end of the time period. As compared to control plasma,
the addition of citrate did not appear to harm the clotting factors. There is
actually some suggestion that the citrate may actually help preserve
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platelets. This would be consistent with the hypothesis that citrate inhibits
some of the proteases.
2% Citrate Plasma
Measurement Day 1 Day 6
Fibrinogen (mg/dl) 243 239
Factor II (%) 104 91
Factor V 49 22
Factor VII (%) 81 72
Factor VIII (%) 103 92
Factor IX (%) 106 95
Factor X (%) 92 97
Platelets (103/10-6L)311 239
PT (sec) 14.1 16.2
PTT (sec) 31.4 47.9
Control Plasma
Measurement Day 1 Day 6
Fibrinogen (mg/dl) 241 239 -
Factor II (%) 103 93
Factor V 58 25
Factor VII (%) 86 69
Factor VIII (%) 100 89
Factor IX (%) 106 92
Factor X 1%) 93 85
Platelets (x103/10'6L)317 192
PT (sec) 13.7 19.5
~PTTw(sec) 32.5 50.2
Significantly, bacteriology experiments showed that 2% trisodium
citrate strongly inhibits growth of Escherichia coii and completely inhibits
the growth of Staphylococcus epidermidis. Growth of bacteria (primarily
skin bacteria from inadequate surface disinfection) in platelet concentrates
significantly lowers the useable life of platelet-rich solutions. Addition of
citrate inhibits bacterial growth thereby potentially extending the life of
such
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concentrates. As has been demonstrated above, addition of citrate does not
damage the plasma constituents and actually significantly enhances the
production of cryoprecipitate. Therefore, it is proposed to significantly
increase the level of citrate in blood collection bags from the 0.4% currently
5 used for anticoagulation to at (east 2% trisodium citrate by weight. This
level would inhibit or kill many contaminating microorganisms and would
render the plasma more suitable for production of cryoprecipitate. It is also
a
simple matter to add trisodium citrate just before cryoprecipitate production
where levels beyond 2% are needed.
10 Added citrate appears to enhance the susceptibility of microorganisms
to a variety of "disinfecting agents" including heat. In one experiment 2%,
sodium citrate was added to a typical bacterial growth broth. Twenty-five
ml aliquots of the broth were spiked with 1 x 104 organisms of either
Escherichia cvli or Staphylococcus epidermidis. Samples of the broth were
15 brought to 2% by weight trisodium citrate and then subjected to
"pasteurization" at 65°C for either 5 or 10 min. after which the
samples
were plated on growth media and incubated. The 10 min citrate treatment
caused total destruction of the bacteria. At 10 min the control bacteria were
essentially unaffected. However, the 5 min treatment citrate did not kill all
of the E, coli bacteria (approximately a 3-log kill). Staphylococcus
epidermidis was more sensitive and was completely killed in the presence of
citrate. Addition of citrate clearly enhances the ability of heat to kill
microorganisms. Further, added citrate appears to stabilize labile proteins
against~fieat denaturation. These results indicate that addition of increased
citrate makes possible effective heat treatment of the plasma.
A problem with platelet concentrates and with plasma is the growth
over time of bacteria that are originally present in very low numbers. Some
of the contaminating bacteria apparently come from the skin surface when
the blood is obtained by venipuncture. Further, there is growing evidence
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that blood is not completely aseptic. That is, there are normally a small
number of bacteria circulating in the human bloodstream. Normal immunity
prevents the overgrowth of these bacteria. To simulate this situation 10 ml
samples of human plasma were inoculated at very low levels ( 10 organisms
per ml) with the bacteria listed in the following table. Either normal plasma
(N) or plasma with 2% by weight of citrate (C) was employed. The samples
were incubated at room temperature for seven days with a sample plated on
growth agar at each time point. Three different human plasmas were used,
but all produced identical results. In the table "ng" - "no growth" while
"+" indicates some bacterial growth and "+ +" indicates more extensive
growth.
a) Escherichia coli
b) Klebsiella pneumoniae
c) Staph ylococcus epidermidis
d) Staphylococcus aureus
e) Pseudomonas fluorescens
f) Yersinia enterocolitica
g) Serratia marcescens
InoculumDay Day Day Day Day Day bay
1 2 3 4 5 6 7
N C N C N C N C N C N C N C
a) ng ng ng ng ng ng ng ng ng ng + ng ++ ng
b) ng ng ng ng ng ng ng ng ng ng + ng ++ ng
d) ng ng ng ng ng ng ng ng ng ng + ng ++ ng
e) ng ng ng ng ng ng ng ng ng ng + ng + ng
f) ng ng ng ng ng ng ng ng ng ng + ng + ng
g) ng ng ng ng ng ng ng ng ng ng ng ng ng ng
At days six and seven, the normal plasma showed growth of all of
the inoculated bacteria species except for Serratia. On the other hand, none
of the plasma samples containing citrate demonstrated any bacterial growth.
This indicates that 2% by weight citrate is able to inhibit strongly the
growth of a wide range of bacteria. Combining these results with the
favorable platelet results demonstrates that addition of 2% or more citrate
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to platelet concentrates can preserve the concentrates .against bacterial
growth without damaging the platelets. If there is any concern about excess
citrate in the platelets, it can be readily removed by treatment with an anion
exchange resin or similar material.
It was suspected that the failure to observe Serratia was due to the
slow growth rate of this organism. Therefore, the experiment was repeated
using Serratia marcescens and Staphylococcus epidermidis to inoculate
plasma samples at the level of 100 organisms per ml. In this case the one
day time point for Serratia showed 92 colonies while that for
Staphylococcus showed 101 colonies for the normal plasma. Thus, no
growth was observed in either case for the citrate-containing plasma.
The precise mechanism by which citrate and similar molecules act is
not know. Multiple carboxyl groups appear important particularly in the case
of cryoprecipitate. Oxalic and lactic acids are less effective. It seems
possible that some type of charge interaction favors the precipitation of the'
clotting factors. As mentioned above, there appears to be good data
supporting the hypothesis that citrate preferentially cross-links the
cryoprecipitate proteins. While chelating ability is clearly important for the
well known anti-coagulation effects of citrate, chelation may not be central
to the present invention as isocitrate is believed to be a poorer chelating
agent than citrate. It may also be that the participation of many of the
effective molecules in the tricarboxylic acid cycle may also be related to
their effects-particularly those on bacterial growth. That is, it seems likely
that the cryoprecipitate phenomenon and the bacterial growth phenomenon
have separate explanations.
The invention has mainly been described with regard to
cryoprecipitate. There are other characteristics, and blood components, and
products that form the subject of the invention. These are illustrated in the
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attached FIGURE 2 that shows a Fractionation Scheme, and different blood
components and plasma proteins, which are obtained from the system and
to which the citrate technology is applied. The starting point is collected
blood-here a bag of CPDA (citrate-phosphate-dextrose-adenine) treated
blood. The extra citrate could be added to this first bag or could be added,
for instance, at the stage of the second empty bag with that bag containing
citrate at an effective concentration of greater than about 2% to about 10%
by weight or by volume trisodium citrate. As mentioned above, the citrate
can even be added at the frees/thaw step. Because of this further addition
of citrate, different new products are obtained.
This citrate-related process has the following features that relate to
FIGURE 2:
1. The products and processes starting from the Second Empty
Bag onwards show improvements in purity and safety over normally
fractionated plasma. The enriched cryoprecipitate 1 is at a higher yield than
in the prior art. The enriched cryoprecipitate 1 has fewer "contaminating"
extraneous serum proteins as compared to "normal" cryoprecipitate. If crude
fibrin glue 4 is produced directly from enriched cryoprecipitate by addition
of
thrombin or prothrombin complex (Factors II, VII, IX, and X), the resulting
glue is superior in strength to crude fibrin glue produced in the same manner
from "normal cryoprecipitate." It compares favorably, and may actually be
superior to, highly refined fibrin glue. The product is safer because citrate
inhibits .bacterial growth and because citrate can facilitate a
"pasteurization"
step to further destroy pathogens. Further, because of the higher yield of
enriched cryoprecipitate, and greater strength of the crude fibrin glue
autologous fibrin glue becomes much more feasible. Autologous products
are inherently safer. Further, because of the much greater yield of fibrinogen
with enriched cryoprecipitate, it is feasible to produce "refined" fibrin glue
or
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19
sealant which is primarily pure fibrinogen plus thrombin (added during
application) although Factor VIII and other ingredients may be included.
The cryo-depleted plasma 3 is safer for the above mentioned reasons.
It is also inherently safer because it contains far less fibrinogen than
normally processed plasma. This means it is virtually impossible for this
material to develop microclots due to activation of the prothrombin
complex-such microclots can cause intravascular coagulation and related
transfusion problems. Also, the elevated citrate level reduces the likelihood
. of activation of prothrombin complex . A second bag with the increased
carboxylic acid and/or citrate derivative (or other use of higher levels of
citrate) is a feature which prior to the present invention has never been part
of a blood fractionation process or part of the production of blood
components for clinical use. There are significant advantages to using
increased citrate (or related carboxylic acids).
2. Fibrinogen 3, Fibrin Glue 4, von Willdebrand's factor 5,
Fibronectin 6 and Factor VIII 7 can be produced by standard
chromatographic methods, but use of differential extraction simplifies the
process. As mentioned above, the enriched citrate cryoprecipitate 1 is
mostly insoluble in water. It is soluble in normal saline and very soluble as
saline to which citrate is added. Therefore, extraction with buffers having
different amounts of citrate results in preferential solubility of the
different
products.
3. Nroducts 8, 9 & 10 can be produced from Single Donor or from
Pooled Plasma (preferably in combination with Iodine disinfection-as
detailed, for example, in U.S. Patent No. 6,045,787).
4. It is also possible to use iodine disinfection before the
cryoprecipitate step. A co-pending application demonstrates that citrate
enhances the iodine treatment. However, the columns used in the
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referenced patent remove citrate. Therefore, after Iodine disinfection,
additional citrate can be added at the second blood empty bag step but
before the freeze/thaw procedure.
Albumin & Globulins (product 10) may be further fractionated to yield
5 separate albumin and gamma globulin. Generally, well-known fractionation
techniques are used. Anion exchange (DEAE) is used to purify prothrombin
complex. DEAE/sephadex can be used in a single donor process both to
purify prothrombin and to dehydrate. Serpines 9 are purified by standard
methods. Yields are improved because of the protease inhibiting properties
10 of the added citrate. Again, these products are safer because of the
citrate,
because of the iodine step, because single donor products can be readily
prepared and because pasteurization is facilitated.
The invention covers the process and products obtained by the
process. The following claims are thus to be understood to include what is
15 specifically illustrated and described above, what can be obviously
substituted and also what incorporates the essential idea of the invention.
The illustrated embodiment has been set forth only for the purposes of
example and that should not be taken as limiting the invention. Therefore, it
is to be understood that, within the scope of the appended claims, the
20 invention may be practiced other than as specifically described herein.
