Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~WO 95126749 2 ~ 8 5 2 2 8 F~ U.. _. . /
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TITLE OF TT~F INVENTION
CRYO~E~ ATED NATIVE FIBRINOGEN CONCENTRATES
Backaround of the Invention
Fibrinogen is one of the numerous proteins of
blood plasma from which the rhonl -r and - ~n; Fm
emanate to form the structure of fibrin clot. Its
10 ubiquitous physiological role in internal restructuring
or repair of tissue discontinuity has been extended to
CUL L e~u..ding role of external application developed over
the past scores of years as a concentrate processed from
plasma f or tissue bonding under such descriptive terms as
15 fibrin clot, fibrin adhesive, fibrin weld, fibrin
sealant, tissue sealant, and so on.
The clinical u6e of fibrin ~ e~a~ed from plasma by
various methods of precipitation and c~h~mir:~l
insolubilization has gradually emerged for such early
20 clinical use6 a6 h~ -_Lyutic adhesive powder with small
opening vessels (Bergel, S., Deutsch. Ned. Wochenschr.,
1909, 35:613-665), as a hemostatic agent in cerebral
surgery (Grey, R. G., Surg. Gynecol. Obstet., 1915,
21:452-454~, in suturing of peripheral nerves (Matras, H.
25 et al., Wien . Med. Wochenschr., 1952, 122: 517-591), and
gradually ~Yr~n-1in~ to the repair of traumatized tis6ue
(Brands, W. et al., World J. Surg., 1982, 6:366-368), and
the anastomoses or restructuring of cardiovascular or
severed cardiovascular, colon, bronchial, nerve endings
30 and other anatomical discontinuities or surgical
incisions to replace or augment conventional suturing.
To such clinical applications, the native fibrinogen
concentration in plasma averages 513 milligrams per
deciliter (mgm/dcl) according to standard ~7 ;nic~l
35 assays, ranging from 229 to 742 mgm/dcl standard
21 85228
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-- 2 --
deviation, ba6ed on the photometric measurements of
turbidity from clotting (Castillo, ~.R., et al.,
ThL '-.i.C, 1989, 55:213-219). This range of
conc~ L~tion cuLL~ u-lds to 0.229% to 0.742% (average of
5 0.513). In a typical prior art example such a6 Dre6dale
et al ., Surgery, 1985, 97: 750-754, the time sequence of
cryofreezing, thawing, and centrifuging produce6 a
fibrinogen cc"~cell~Late of only 2.16% t2160 mgm/dcl). The
resulting fibrinogen ~ullc~l.LLdtes typical of the prior
10 art are too dilute for practical clinical needs owing to
inordinately low viscosity, very much like that of water,
at room operating temperatures. This product require6
standby ~ h; 11 i n~ re6ulting in lack of viscous
adhesiveness needed for manageable and effective surgical
15 anastomoses or approximating of tissue incisions.
Conventional plasma products are dialyzed, heat
inactivated, delipidized, filtered, and/or irradiated.
None of the prior art provides the essential
descriptive details on productivity and efficiency, and
20 product qualifications with supporting tests for
manageability, viscosity, adhesion, and effective high
solids fibrinogen .:oncel,~Lates for 6urgical
reconstitution of severed or incised tissue.
One conventional means for separating or
25 conc:~:n~Lating fibrinogen from ~ 1 ;~n plasma is by
various chemical precipitating procedures of ~lm~rtllres
with cu-~ct-,~L~ted salt solutions, such as semi-saturated
60dium chloride, 6aturated ammonium 6ulfate, and by cold
ethanol and other low molecular weight organic compound6,
30 notably amino acid6 6uch as glycine, and uu~
combinations thereof. These chemical precipitating
procedures lmposing varying degrees of denaturization in
contrast to the non-chemical cryoprecipitation methods
which are applied to the preparation of high purity,
35 single fibrinogen entities stripped of the nascent,
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natively a6sociated symbiotic plasma proteins which may
remain dissolved in the added chemical precipitants. The
na6cent, hereinafter termed native, proteins include
glycoproteins of various conf igurations with carbohydrate
5 structures in their derived acetylated and aminic forms.
Their presence have been in many instances purposely
discarded in the course of the chemical precipitive
preparations of fibrinogen, but now have been discovered
to impart signif icant adhesive tensile strength in the
10 mea~ uL Ls of the bonding strength described in the
various examples in the present invention.
A cryoprecipitate of native fibrinogen heretofore
has not been generally reco~n; 7~-d as a preferred source
of ~nh;~nr~cl high solids fibrinogen concenLL~tes.
15 Associated native mucoproteins which lend viscous tissue
adhesive qualities have been removed from conventional
products by chemical precipitation. Fibrinogen stripped
of the associated mucoproteins is also routinely
prepared. Such adventitious rh~-nir~l stripping imposes
20 major physical conformational changes in the molecular
form and shape of the native fibrinogen structure that
may lead to denaturing or depolymerization on storage, in
turn affecting the desirable initial viscous tissue
bonding quality for reliable expected clinical
25 performance.
Cryoprecipitation imposes ~LU~;LUL~l changes in
the plasma proteins due to fibrinolysis during the
prolonged cryogenic state of conventional methods in
terms of the inevitable t ~LUL~ time k;n~tjrc The
30 prior art has disclosed the use of a wide range of
t~ L~Lulc:-time variables but provides no indication of
the effect of varied temperature-time kinetics on
productivity and product quality on the f irst procedural
step of cryofreezing. Rather, the prior art literature
35 infers that longer periods of cryofreezing and thawing is
WO 95126749 2 1 8 5 2 2 8 ~ ~ " " ~ 1487
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n~ c6;-ry for attaining higher purity of the fil:rinogen.
For instance, the clinical preparation -nc F-c by
.yur~zing at -80C specified for at least 6 hours
(Gestring, G.F., et al., 1982) later this was increased
5 to at least 12 hours (Dresdale, A., Surgery, 1985,
67:751); and again later for at least 24 hours (Spotnitz
et al., The American Surgeon, No. 7, August, 1987). None
of these rl;niCi~1 preparations provides data on
productivity and qualif ications of the resulting
10 cryoprecipitated products in support of the need for
increasing the stated u.yur.~eGing time.
On the contrary, as indicated in Application
Serial No. 07/562,839, Example I, Table 1, Fraction I, in
terms of product gram yields, rather than increasing the
15 cryofreezing time from 6 to 24 hours, it was discovered
that as little as 1 hour is adequate with substantially
the same gram yield as with four hours of cryofree2ing.
Apart from reflecting a substantial increase in process
efficiency, this reduction in :LyurLeezing time makes the
20 process and product suitable for urgent need of
autologous clinical E Ll:paLc.Lions. Obviously, for
immediate autologous clinical usage for ready surgical
av~ hility only one hour of the prolonged ~:-yùrL~ezing
times is proven to be highly acceptable. In none of the
25 prior art descriptions has there been any consistent
indication of both the col.c~"L-clte yield and the product
characterization and quality testing.
Following the cryoprecipitation process step,
thawing is the next essential n,~nt of the process
30 during which the solid heterogeneous crystalline-like
frozen mush is transformed into two phases of sedimented
precipitate and a viscous fluid with a glacialized
h~ J~l~euus solid plug of ice, hitherto not rF-~-o~n; 7~d in
known prior art. With prolonged thawing, either as the
35 usual separate step or simultaneously during
2 1 85228
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centrifugation, the ice ~uyLes6ively melts during the
thermal drift along with concomitant re-dissolving of the
plasma proteins. The control of the thermal drift from
cryofreezing to the completion of thawing is critical to
5 the resulting concentrate yield, the solids
cul.ctl.LLe~tion, and the distribution of the uus
associated plasma proteins through the solidus - liquidus
equilibrium transition t~ ~Lu- ~ depicted as follows:
Process phases
10 cryoprecipitation ----> thawing ----> centrifugation
OC
(solidus~ (de-icing) (liquidus)
wherein the frozen solid plasma releases the
15 cryoprecipitated insoluble f ibrinogen and relatively
soluble associated proteins which are important for the
fibrinogen col-ccllL~eltes in tissue bonding and controlled
to desired contents in the cul.~ellL- Ites. The ratio of
the f ibrinogen associated proteins thus can be regulated
20 by the thermal drift of the solidus to liquidus
tran6ition as the more soluble associated proteins re-
dissolve in time. The plasma proteins serve as endowed
bioadhesives, characterized as mucoproteins and
chemically known as glycoproteins, which are indigenous
25 to the fibrinogen and also intended to be retained as
much as possible by the temp~ u- ~ time thermal drift
control of the process of the present invention. The
thawing is readily evident from the progress and extent
of measured de-icing in turn regulated by selected time
30 at tP ~ Lu~ ~ for any required retention for the
adhesive quality in tissue bonding. The retention of the
associated plasma proteins is highly llepPntlPnt upon the
thermal drift from the cryogenic state through the icing
equi~ibr~ with minimal time in the liquidus watery
21 85228
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phase during which the a660ciated proteins begin and
continue to re-dissolve from the cryoprecipitated state.
The thawing time in numerous known, pl-hl i ~:h~
methods is not consistent and in no instances correlative
5 to either the quantity or quality of the attained
fibrinogen concentrates. For instance, the specified
thawing time varies from such indefinite t tlLu~t time
kinetics as at 4C "when liquid" (Gestring, supra); at
4C "for several hours" (Dresdale, supra); and at 1C to
10 6C for 20 hours (Siedentop et al., Laryngoscope, 1985,
95:1075); in no instances of this prior art is there any
indication of the gram yield, solids content, or
qualification tests for effectiveness. In all these
cited instances, the prolonged thawing leads to re-
15 dissolving of the cryoprecipitates during thetemperature-time thermal drift with inordinate 1055 of
f ibrinogen and its associated proteins with solids
contents ranging from as little as 39~ to 696. The need
for minimizing the temperature-time thermal drift was
20 made evident in Application Serial No. 07/562,839, by
which the products of this invention have been produced.
Following thawing, the ~:Ly~ cipitate is
subjected to physical separation by centrifuging at
specified gravitational (xg) force in the course of the
25 t~ ~lLu.~ time thermal drift. As indicated in the
preceding references, centrifugation involves a wide
range of speed (RMP), gravitational force (xg),
temperature, and time. These include, for instance,
llnl:peci fied cold centrifuge at 2300 xg for 10 minutes to
30 15 minutes (Gestring, supra), 1000 xg for 15 minutes
(Dresdale, supra), 5,000 rpm (lln~r~ri~ied xg) at 1C to
6C for 5 minutes (Sidentop, K. H., supra), and at 6500
xg for 5 minutes at 4C (Spotnitz, supra), again with no
indicated productivity and q--;l;flc~tion tests.
2 1 85228
~Vo 95l26749 I
*7
Given the variety of the procedural details for
producing fibrinogen concentrate for surgical use in
tissue bonding, the prior art provides no cogent criteria
of consistent productivity from plasma with measured
5 criteria of quality for safe, effective and reliable uses
in surgical tissue bonding to which the products of this
invention are directed.
rv of the Inventign
The present invention is directed to a
10 cryoprecipitated f ibrinogen concentrate of native
; An plasma comprising about 6% to about 44% solids
content, wherein about 5% to about 95% i5 clottable
fibrinogen, said cryoprecipitate having a viscosity of
from about 80 to about 430 centipoises, and a tensile
15 break force of about 1 to about 81b-f/in-w.
The invention is directed to a ~LyopL~cipitated
native, und~l.atuL~d, non-lyophilized fibrinogen
.,I...ellLL~te. The fibrinogen col.c~llLLclte of the present
invention may be associated with nascent indigenous
20 proteins which enhance the viscous adhesion in tissue
bonding .
Another objective is to provide a high solids
f ibrinogen concentrate as versatile f ibrin sealants
amenable to a diversity of ambient thrombin, direct
25 thermal, and spectrally induced thermal ab60rptive
bonding in a broad range of f ibrinogen/protein ratios .
A still further objective of the invention is to
provide test methods for effective tissue bonding for
qualification of the native fibrinogen concentrates for
30 use in surgical applications.
A native, undenatured high concentrate autologous
concentrate sealant in minimal processing time for use as
sealant in emergency surgical needs is herein provided.
- 21 85228
WO 95/26749 I ~IIIJ~ ~987
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A still further objective is to determine and
utilize the composition of the f ibrinogen protein
cryoprecipitated products obtained by a series of
y1oule~ive recycling of the 1ecuveLed supernatant plasma
5 serum.
Detailed DescriPtion of the TnYentiOn
The present invention is directed to a
cryoprecipitated f ibrinogen concentrate of native
liAn plasma comprising about 6$ to about 4496 solids
lO content, wherein about 5% to about 95% is clottable
fibrinogen, said cryoprecipitate having a viscosity of
from about 80 to about 430 centipoises, and a tensile
break f orce of about l to about 8 lb-f / in-w .
A tissue adhesive comprising a cryoprecipitated
15 fibrinogen concentrate of ~ l i~n plasma comprising
about 6% to about 44% solids content, wherein about 5% to
about 95% is clottable fibrinogen, said cryoprecipitate
having a viscosity of from about 80 to about 430
centipoises, and a tensile break force of about 1 to
20 about 8 lb-f/in-w is al60 within the scope of the present
invention .
The f ibrinogen concentrate of the present
invention comprises a solids content made up of
~s in the molecular weight range of from about 18
25 including electrolytes and salts; to about 8,000 to about
600,000 Daltons including fibrinogen, and associated
amino acids and proteins including and not limited to
albumin, mucoproteins, Factor XIII, fibronectin,
rl~-~innqon, prothrombin, thrombin, other proteins
30 including and not limited to growth factors, and the
like. The r~~-ininq 94% to 569~ of the concentrate is
water and other liquid components of plasma. The
transition temperature Yaries f or each of the c ,ent
parts of the solids content of the fibrinogen
21 85228
Wo gs/26749 F~I1~,., /
_ g _
concentrate. As the thawing time decreases, residual
icing increases.
The cryoprecipitate of the present invention may
originate from mammalian plasma including and not limited
5 to human, bovine, porcine, rabbit, eovine, and equine
plasma. Human plasma for use in the present invention
may be allogenic or autologous. Any of the mammalian
plasmas may also be cryoprecipitated together with any of
the associated proteins identified above. For example,
lO albumin may be combined with plasma, wherein the ratio of
plasma to albumin is from about 100:0, 90:10, 80:20, and
60:40. Further, the supernatant ~ ining from plasma
which has been passed through one cycle of cryofreezing,
thawing, and centrifuging, may be reused or recycled to
15 further prepare a ~:~ y~L~cipitate therefrom. Pooled
cryoprecipitates from several sources and/or processes
are also within the scope of the invention. Pooled
cryoprecipitate may also result from various processes or
repeated processes with variations in time, ~ ~ atuLe,
20 cryofreezing, thawing, and centrifuging.
?' 1 i;7n plasma or cell plasma and modifications
thereof with supplemental additions and ; nrll7cinnc of
various kinds are within the scope of the present
invention . Modif ications and/or supplemental additions
25 may include coprecipitants that induce precipitation of
associated plasma proteins, or similar molecular or
biologically active entities, for conversion of the
concentrates to new products f or specif ic uses . These
include such common modifications as (a) anticoagulants
30 including ACD citrate or heparin, (b) anti-fibrinolytic
agents such as aprotonin, and epsilon-;minnr~rroic acid,
(c) coagulating agents such as calcium - 'q, (d)
viscosifying and thixotropic -- - '; fi~rs either naturally
occurring or synthetic polysaccharides,
35 mucopolysaccharides, and polygalacturonic acid, (e)
21 85228
WO 95/26749 I ~ ~
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bioadhesives in the form of mucoproteins or glycoproteins
indigenous to plasma or serum or their synthetic analogs,
(f) surfactants 6uch as naturally occurring - ~ ul eins
~nd mucopolysaccharides of the N- or 0- substituted
5 neuraminic acids, (g) supplementing with precipitating
agents affecting the electrolyte balance and/or
osmolality of the plasma such as ethanol, urea, glycine,
and their homologous chemical structures, and (h)
preservatives or choice against bacterial or
10 microorganism activity.
Supplementation of the f ibrinogen protein
concentrate may be carried out in several ways, including
and not limited to adding supplements to the initial
plasma and then processing them together or by
15 supplementing the fibrinogen cul.~el~LLc~te after
processing .
In order to establish productivity, def ined
qualification tests, and standards lacking in the prior
art, a novel and more ef f icient process engineering
20 system was devised as herein described. A substantially
higher level of solids content within the f ibrinogen
L~te was achieved by applying a controlled thermal
drift throughout the integrated uLyu~L-acipitation~
thawing, and centrifuging steps. In addition, the
25 overall processing time is also shortened over that
described in the prior art. The qualif ications provided
by the product of the present invention, serve as a basis
for specifications for clinically safe and effective
~nhAnr ed fibrinogen concentrate products for large scale
30 production from pooled plasma and for limited small scale
lots of t~nh~nced autologous fibrinogen _c,l,c~:l.l L~tes from
patients in view of the prevalent risks of viral
infections, notably numerous forms of hepatitis and human
-~ficiency virus (HIV), from pooled or single-donor5 sources .
21 85228
,. ~
~WO 9C126749 P~l/u.,,~,r~'t87
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The process for producing a fibrinogen .:v.lc~llLL-te
from 1 ;An plasma comprising freezing said plasma to
- a temperature of at least about -20c for less than four
hours, thereby producing frozen plasma; thawing said
5 frozen plasma at a temperature of at least about 40C for
a time sufficient to attain from about 5% to about 95%
residual icing, thereby producing thawed plasma; and
centrifuging said thawed plasma to produce a fibrinogen
vvllct:llLLclte having about 6% to about 44% solids content
10 is also within the scope of the present invention.
The freezing step of the present lnvention
subjects the plasma to a temperature below the freezing
point of the plasma. The temperature for freezing
1 ;An plasma in the process of the present invention
15 is at least -20C, more preferably about -20C to about -
120C, even more preferably -80C. Freezing may be
performed for less than about four hours, more preferably
about 0 . 5 hours to about 4 hours, even more pref erably
about 1 hour. At these temperatures, the plasma is
20 frozen to tempelclLuLes which compact the cul.cellLLc.~e
thereby making it more dense.
Thawing for purposes of the process of the present
invention takes place at a temperature of less than about
room t~...~eLClLULe, more preferably from about 4C to about
25 10C, even more preferably about 4C. The thawing step
of the present invention is performed for a time range of
f rom about one hour to about 3 o minutes . The time and
temperature of the thawing step of the process of the
present invention are performed such that from about 5%
30 to about 95% residual icing, more preferably about 30% to
about 95% residual icing, even more preferably about 30%
to about 40% residual icing is formed.
For small lot clinical preparations of blood
fractions from single donor lots of blood, the
35 ;, yv~rl:cipitates are thawed and then centrifuged to
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separate specific fractional products generally in a
closed system to avoid environmental contamination using
blood collection kits in conf ormity with standards
prescribed by the American Association of Blood Banks.
5 In bulk larger scale processing of pooled lots of blood,
~.u~.iate form and mean6 of cont~i t against
enviL ~11 Qontamination are likewise provided for in
similar processing stages or steps of u.yo~L~cipitation,
thawing, and centrifugation. Containers for use in the
10 preparation of the cryoprecipitate of the present
invention preferably have a surface:volume ratio of about
4.38:1 to about 1.65:1 reciprocal centimeters (cm~l).
Cryoprecipitation is used as the initial,
principal means of separating fibrinogen in preference to
15 the previously mentioned precipitation alternates. It is
m~nir~lly chemically reactive or disturbing to the
intricate native conf iguration of the plasma proteins .
Unlike lyophi 1 i 7ation, cryoprecipitation does not remove
water from the plasma. Accordingly, IIYdLOg~:rI bonds are
20 not broken in the c;Lyu~.~acipitate and thus, water need
not be added to reconstitute it as is required by some
conventional products. Surprisingly, cryoprecipitation
in itself has not been adequately explored or studied in
the kinetic temp~ Lu.~ time precipitation along with the
25 thermal drift in relation to cooling rates and thawing
rates. Considering the presence of ~lu..d- ~ds of protein
~ s in plasma of varying rates of insolubili2ation
to the cryogenic temperature and the reverse of re-
solubili2ation, the thermal drift in each direction
3 o provides the arena f or limiting the nature and
constitution of the f ibrinogen concentrate.
Cryoprecipitation is therefore a preferable means
of producing the f ibrinogen concentrate over the
alternative of precipitation with adjunctive non-
35 physiological chemical precipitants such as saturated
21 85228
~¦IWO gs/26749 P~~ 987
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salts, low molecular organic fluids or organic, ~c
that are 6uspect of imposing major physical,
conformational changes in the molecular form and shape of
the fibrinogen structures (Doolittle, 1975a). Although
- 5 cryoprecipitation i5 not in itself without some
imposition of structural changes, it can be reasonably
conjectured that the transition to and from cryogenic
t~ ~UL~'S through the icing stage under restrictions
of the temperature-time kinetics avoids the potentially
10 drastic chemical environment on the extremely sensitive
Ant chain linkages and their resistance to
fibrinolysis (Doolittle, 1975b).
However, as is generally the inevitable rhPn n nn
of continued rhDmicAl activity in the cryogenic state
15 (Fennema, 1982) of native proteins, particularly that of
associated enzymes and possibly fibrinolytic activity,
the unduly prolonged cryogenic state in terms of the
kinetic temperature-time factor has not been defined in
the conventional practices of preparing fibrinogen
20 c~,..ce,.~L.ltes. A critical feature of the invention is the
discovery of ~Lyu~ecipitation and its hitherto
nonobvious effects on yield and -r-- s of the
cryoprecipitated fibrinogen c.,..ce.,~ tes. None of the
currently available methods and preparations are
25 acceptable for immediate clinical autologous usage within
2 to 4 hours.
Following the cryoprecipitation process step,
thawing is the next essential and critical t of
the process of preparing the cryoprecipitate of the
30 present invention. During thawing, the solid
heterogeneous crystalline-like mush transforms into two
phases of a viscous fluid with a gl~ li7pd homogeneous
solid ice progressively melting with the thermal drift at
thawing. The thermal drift is critical to the
35 c~,..cellLLclte yield, the solids concentration, and the
21 85228
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-- 14 --
distribution of the numerous proteins through the 601idus
- liquidus equilibrium temperature. During thawing/de-
icing, the frozen solid plasma relea6es the insoluble
f ibrinogen and innumerable associated proteins that are
5 important f or f ibrinogen ~u~ L cltes or more properly
termed f ibrinogen protein concentrates tiP~Pn~l i n~ upon the
attained purity of the f ibrinogen concentrates . The
latter is the ratio of the f ibrinogen as60ciated proteins
which can be regulated by the thermal drift of the
10 solidus to liquidus transition as the more soluble
associated proteins re-dissolve in time. These include a
range of bioadhesives (Gurny and Junginger, 1990)
characteristic of the _:~ ~c,teins, chemically known as
glycoproteins which are indigenous to f ibrinogen, and are
15 also intended to be retained as much as possible within
the purview of the present invention. The thawing is
readily evident from the ~LOyL~:SS of de-icing and thereby
regulated by ~lPcted time and temperature. Thus,
thawing retains the useful and/or valuable plasma
20 proteins natively associated with the complex structures
of f ibrinogen .
By applying a specif ied critical control at the
de-icing or thawing stage of the solid cryoprecipitate to
the liquid watery state, throughout the time of continued
25 thermal drift to and from cryofreezing, the new
processing system results in considerably higher yields
and solids content of the f ibrinogen protein concentrate
with a diversity of the associated useful protein
contents. Thermal drift refers to the temperature
30 differences between the external thermal exposure and
internal thermal plasma states during the three
procoC:sinq stages of cryoprecipitation, thawing, and
centrifugation. The overall process efficiency is
thereby markedly and unexpectedly increased and
Wo 95l26749 2 1 8 5 2 2 8 P~l/u~ ,
-- 15 --
processing time considerably shortened from the starting
plasma to the separated fibrinogen ~OI~Ce:l~LLclte.
The retention of the associated proteins is highly
d~p~ntlAnt upon the thermal drift from the cryogenic state
5 through the icing equilibrium to centrifugation by means
of minimal time in the liquidus watery phase at which the
associated proteins begin and continue to dissolve.
Following thawing, the cryoprecipitate is then
subjected to physical separation by centrifugation as the
10 continuation of the temperature time frame of thermal
drift but with the minimal centrifugation time frame and
with specif ied gravitation force at stated revolutions
per minute (RPM). As discovered for this application and
indicated hereinafter, the need for minimizing the
15 temperature time thermal drift presents a critical
process int~ - ' i Ary to assure maximizing the yield of
the clinically useful fibrinogen by the most minimal
thermal drift possible to which this application is
directed .
Solidus-liquidus eguilibrium transition
t~ UL ~ is a temperature at which, f or each -nt
of the solids content of the f ibrinogen concentrate, the
solid (i.e., ice or frozen) phase and liquid phase of a
component are in equilibrium. For example, the solidu5-
25 liquidus equilibrium transition t ~tUL~: for water i5
the temperature at which ice and liquid water exist in a
percent ratio of about 50:50. R~5~ 1 icing refers to
the amount of ^nts in the solid phase, i . e., ice,
as compared to the components which have passed through
30 the transition temperature into the liquid phase.
Thawing permits each of the nt parts of the plasma
to reach a transition temperature such that the
c~ ts pass from the solid phase to the liquid phase.
By controlling the solidus - liquidus transition with
35 time and temperature in the thawing step, the residual
21 85228
WO 95l26749 .
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icing is thereby controlled. Control may similarly be
established where thawing and centrifuging occur at the
same time.
R~ci~ l icing appears in the form of ice. In
5 examples set forth herein, test tubes were used as a
cont~; L such that residual icing formed as ice plugs.
Residual icing = weiqht or volume of ice ~luq x 100
weight or volume of initial plasma
Following ~l~t~rm; n~tion of the weight of residual
10 icing by weighing, the percent residual icing may be
readily estimated visually. Visual estimation proved
workable in Table 2 below in a range from 10% to about
100~6 residual icing.
Centrifugation is performed to produce a
15 f ibrinogen cc,nce.~ te having about 6% to about 44%
solids content, more preferably about 24% solids content
and even more preferably 12% solids content.
Centrifuging may be performed at a gravitational force of
about 1450xg to about 8000xg, for about one hour.
2 0 EXAMPLE
This Example ~ LLcltes the superior
productivity, process efficiency, and product
qualifications in the ~nhAn~e~d fibrinogen co~.c~llLL~tes of
the present invention . The new and ~nh;`nred f ibrinogen
25 concentrate products, referred to as Product 8, are
produced by controlled t~ ~L.., . time thermal drift
through the solidus - liquidus equilibrium transition
from cryoprecipitation to centrifuged concentrate as
9~Rrr;hed in Application Serial No. 07/562,839. The
30 resulting products, summarized in Table 1, are provided
with essential specif ications and test methods hitherto
not made known or available by the prior art, for assured
safe and effective standards for r.l ;n;C;-l applications.
A conventional fibrinogen product, as disclosed by
2 1 8 5 2 2 8
WO 95l26749
-- 17 --
Dresdale, A., Surgery, 1985, 67:751, is represented by
Product A.
Product A.
Four aliquots, 40 ml each, of fresh frozen plasma
5 were cryofrozen at -80C for 12 hours followed by thawing
at 4C for 4 hours, and centrifuging at 1000 xg for 20
minutes (0. 3 hour) in an International Refrigerating
Centrifuge, Model PR-2. The total lapse processing time
was 16 . 25 hours . The cryoprecipitate was separated from
10 the supernatant fluid layer and assayed for productivity,
evaluated for process efficiency, and tested for
qualifications for bonding strength as indicated in Table
1.
Product B.
Using four aliquots, 40 ml each, of the same
initial plasma as in the preceding Product A, the
temperature-time thermal drift schedule similar to that
of Example I of Application Serial No. 07/562,839, was
applied. The time of ~LyurL?ezing was 1 hour and slow
20 thawing was performed for 1 hour at 37C, followed by
centrifuging at 1000 xg for 20 minutes (0. 3 hour) . The
thermal drift during the thawing and to the end of
centrifuging was thereby controlled to residual solidus
icing thereby minimizing the re-dissolving and loss of
25 the valuable associated plasma proteins into the liquidus
phase .
A comparison of the two respective f ibrinogen
co~ e products is summarized in Table 1 ref lecting
the uus distinctive and surprising features of
30 superiority of the c~ lc~ L~te Product B over that of
prior art Product A with substantial advantages in
productivity, process efficiency, and product
21 85228
WO 95~267q9 1 ~ . IQ~987
-- 18 --
qualifications for surgical tissue bonding are evident in
Product A.
Productivity and Efficiency
Product vields - Solids Assav.
Table 1 summarizes and compares the productivity
in terms of dry solids of the controlled thermal drift
Product B fibrinogen cu..~e~ILl~te with that of the typical
prior art Product A on a ratio (B/A) basis. The
controlled thermal drift Product B provided a concentrate
10 yield 3 . 3 times greater than that of Product A, a solids
content of the concentrate 2 . 0 times higher, and dry
solids 4 . 5 higher.
Clot~hle Fihrinoqen A55aY. ,
This assay of productivity is of prime importance
15 as a qualification for effective and reliable surgical
tissue bonding f or several reasons . First, the assay is
a def initive item of product specif ication related to
controlling the thermal drift from cryoprecipitation to
centrifugation. Second, the assay affects and relates to
20 preemptive h~nrll in~ quality in terms of measured
viscosity and the effectiveness of the inherent, primary
adhesive quality in rejoining cut, severed surfaces by
adhesive bonding with a prototype ex anima tissue such as
chamois. Third, the assay includes the nonclotted native
25 protein ~ ~s of the fibrinogen ~ CtlILL.lte as a
measure of the retained native bioadhesive glycoproteins
and numerous other valuable hematological factors, cell
growth factors, and the like.
Two methods of determining clottable f ibrinogen of
3 0 the concentrate products were used:
1. The clinical photometric measurement of
turbidity of well-dispersed clotted f ibrinogen induced by
the conventional Ellis-Stransky thrombin-calcium chloride
` 2 1 8 5228
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-- 19 --
activation. This assay is useful for relatively low
plasma levels of f ibrinogen adaptable to high solids
cul~cel-~ldtes by serial dilutions within the limits of
accuracy and precision of the photometric sensitivity;
2. A method more appropriate to attaining the
combined assay of clottable f ibrinogen and its natively
associated, extensive range of diverse non-clotted
proteins, by simple difference from the percent solids
assay, is by chemical precipitation using either a non-
10 polar diluent, such as ethanol and the like, or an
electrolyte diluent, such as saturated ammonium sulfate
and the like. The non-polar ethanol precipitant, 4-16
ml/gram ~v-.ce--L~ ate, was used in this and other Examples,
in at least two serial washes of the precipitated clotted
15 fibrinogen concentrates. The washes were then vortexed
to disperse the aggregate clots, centrifuged to firm
S~9ir ~dtion, decanted, and finally dried to constant
weight at 80C, usually in one hour, as described in
Application Serial No. 07/562,839.
The results shown in Table 1 d LLate the
~nhAnrPrl clotted f ibrinogen yield of Product B compared
to that of Product A by a ratio of 4.3/1. As will be
evident in ensuing Example II, as a preferred P~hQrl; t
of Product B, still higher yields of the clotted
25 fibrinogen were attained and PnhAnr~d by higher ratios of
clotted fibrinogen and residual plasma protein in the
f ibrinogen coo~ LL ate .
Residual Proteins Assay.
This assay is based on the difference between the
3 0 dry solids yield, Table l, and the clotted f ibrinogen
yield. The assay constitutes all of the cryoprecipitated
proteins and all other residual, symbiotic molecular
organic and inorganic electrolyte constituents, including
residual calcium which is available and proven sufficient
` 21 85228
W0 95/26749 r~
-- 20 --
for thrombin polymerization of the fibrinogen to fibrin.
The results in Table 1 demonstrate superior residual
protein yield with a ratio of 5 . 8 /1 attained with the
Product B compared to that of Product A.
5 Product ef f iciencY .
Taking into consideration the important proc~cs;n~
~n~;n~ ~ing ~ of time, Product B was prepared in
approximately one-seventh elapsed time (1/17.1) of
Product A.
10 Oualif ication Testinq
Of equal importance comparable to productivity and
process efficiency is the qualification testing of
essential product performance features such as ea6y
h;~nrll ;n~, di5pensing, and effective adhesive bonding of
15 the fibrinogen concentrate and making full use of the
native plasma mucoproteins, glycoproteins, and the like
for maximal tissue adhesion. To qualify for those
essential product performance features, 6everal special
preemptive qualif ication tests were devised as indicated
20 in Table 1. Preemptive refers to testing for measured
physical constants, notably viscosity and adhesive
bonding using ex vivo coll A~n chamois substrate model in
order to qualify for ensuing in vivo animal testing.
ViscositY Test.
The relatively low solids contents of fibrinogen
c~ el~L,~Ites produced by the prior art, in the range of
3~ to 6%, lack adequate viscous adhesive tenacity. This
is due to watery and unmanageable consistency of the
prior art products at ambient room temperatures from ice-
30 chilled, slightly thickened state. Despite published
clinical reports referred to herein, the lack of viscous,
- 21 85228
Wo ss/26749 1 . 1, ., . _ , .
-- 21 --
adhe6ive tenacity accounts for the lack of clinical
acceptance of f ibrin sealants .
A novel and practical means f or measuring
viscosity of the :Ly~ ecipitates was devised relative to
5 glycerol at 999c wt (1150 centipoises, at 21 + 0.2C, see
Hodgman, C.D., et al., Handbook of Chemistry and Physics,
page 2197, Chemical Rubber Publishing Co., Cleveland, OH,
1959. ) using a standard 20 gauge (G) 1 1/2 clinical
syringe by measuring the applied dispensing force and
10 smoothness profile, recorded in a tensiometer. As shown
in Table 1, the controlled thermal drift Product B with a
2/1 ratio of solids content over Product A increases the
viscosity to even a higher 3.1/1 ratio of ~nhAnc
viscosity .
15 Bondinq strenqth.
A preemptive in vitro qualification test was
devised f or testing the native bonding quality of the
fibrinogen ..,..c~:-,LLclte emanating from the associated
native plasma bioa~h~cives~ notably the chemical variants
20 of glycoproteins. As a collagen bearing substrate of
animal nature, the readily available chamois was chosen.
Chamois, even though chemically processed of all
biocellular components, contains a considerable amount of
viable peptide proline and l.ydL~cy~oline binding sites
25 available for either passive adhesion or thr~r~l ly
energized bonding. The distinction between passive
adhesion and thermal adhesion or spectrally energized
bonding, is not nPr-~cs Irily exclusive but functionally
cooperative .
Passive adhesion, sometimes referred to as direct
adhesion, is the direct attachment exerted by molecular
attraction between the surfaces of two different
materials. This attachment may have a consequent sequel
of chemical, physical, or mechanical r--h~ni! - of
21 85228
Wo 95/26749 1 ~l,I 'A~987
-- 22 --
measurable adhesive strength, ~n terms of force, and
extension to failure or rupture. Passive adhesion is a
particularly important quality in biomedical applications
f or initial tissue adherence of f ibrinogen which in a
5 pure state of itself is non-adherent on the scale of
contact adhesion.
Spectrally energized bonding is the att li -nt of
measured bonding strength (force and extension) resulting
~rom the application of absorbed energy, externally
10 convected or internally induced (including the broad
spectral frequency from simplest thermal heating to
microwave, ultraviolet, and direct or dye-assisted
absorptive laser energy) . Generally, the th~rr-l ly
energized bonding is the result of specific or varied
15 --^h~ni~ of molecular removal of water, carbon dioxide,
ammonia, and the like, through a myriad of possible
inter- and intra-molecular reactions. Often, passive
adhesives involve progressive interactions with the
substrate by slow or delayed intramolecular reactions
20 thereby elevating a proportionate share of the adhesive
strength approaching the strengths of energized bonding.
The preemptive d~p~nrl~nr~e on the ex vivo chamois
on tenslle bonding testing, before undertaking in vivo
animal testing, is fortuitous on several accounts for
25 evaluating quality of the bonding strength of fibrinogen
concentrates from different donor plasma and
modifications in processing steps. The chamois adhesion
and bonding is a rapid test done in a matter of several
hours, compared to days and months in animal tests. It
30 provides practical evaluation in terms of tensile break
force, a broad range of static and dynamic testing, and
~S6D~ L of the nature of energetic bonding and direct
adhesion. The chamois testing is considerably less
expensive than animal testing, and capabl~ of reasonable
-
2 1 8 5228
Wo gs/26749 ~ 987
-- 23 --
correlation to animal testing. Hence ~lPp~n~ re on in
vivo animal testing is thereby decreased.
The ex vivo chamois bonding test is carried out in
a manner similar to the conventional in vivo thrombin-
5 calcium activation of f ibrinogen to f ibrin . Three inchlong by half-inch wide strips were cut laterally at the
mid-point. These strips were rejoined at the cut with
f ibrinogen concentrates f or the f irst stage of viscous
adhesive bonding by the associated mucoproteins. The
10 fibrinogen concentrate was applied as thin bead extruded
from a 1 ml syringe through a 20G 1 1/2 needle to the
laterally severed edges of the chamois strip pretreated
with l to 2 drops (0.025 to 0.05 ml) of solution
comprising 5 to 25 NIH units of thrombin in 40 mM calcium
15 chloride per one ml of f ibrinogen concentrate .
The rejoined mid-cut butted edges of the test
strips were placed in a 100C oven for 30 minutes to
activate maximal thrombin bonding, then cooled to room
temperature for at least 30 minutes before testing for
Z0 tensile bond strength. The bonded chamois test strip was
inserted lengthwise between the tensile grips of a
tensiometer provided with chart-recorded force on
continuous straining to the rupture of the bonding. The
Instron Tensile Tester, Model 1130, was used in the
25 tensile straining of a test length of one inch interposed
lengthwise between the grips so that the rejoined bonded
butted edges of the chamois specimens were set precisely
in the middle between the grips. The tensile straining
was at the constant cross-head speed of 20 inches per
30 minute. Similar cpe~i~ -, having two pieces of chamois
bonded by the cryoprecipitate of Product A and thrombin
were prepared.
The resulting tensile bonding strength was
det~rm;n-~d from the dimensions of cross sections of the
35 bonded transverse area of the cut half-inch width
21 85228
Wo gs/26749 E~
- -- 24 --
multiplied by the measured thickness of each chamois test
spef 1r-~1 which varies from 0. 018 to 0. 032 inch. Two
options are available: first, in terms of the pound-force
to bond failure, break or rupture per inch width,
5 ~ ssed as lb-f/in-w and second, as the C~LL_~LJnl~fl;nfl
ultimate tensile stress in pound per square inch,
expressed as lb-f/sq in. The first option as tensile
break force per unit inch width, is preferably provided
in each Example as the indicated force is more readily
10 sensed ~ualitatively of the two. The corresponding
elongation to bond failure is also provided in the
Examples. Finally, in order to evaluate and compare the
effectiveness of the variously applied bonding sy6tems in
restoring the bi -~hAnical integrity, the tensile break
15 force and elongation data, as the averages of four tests,
are further provided with the percent regain (sic
restoring) to that measured on the control intact stock
material as is done similarly in in YiVo animal tests.
The results summarized in Table 1 clearly
20 fl LLclte the exceptionally superior bonding strength
with the f nh~nf f A solids content of the fibrinogen
~ .c~ te p~udu~d by controlled thermal drift of
Product B by a ratio (B/A) of 6.8/1 over that of the
prior art Product A; also, Product B provides a
25 superiority of elongation to break by a significant ratio
(B/A) of 2.1/1 over that of Product A. Still further
f nhAn~ ts are made evident in sllrcef~ nfJ Examples of
the pref erred ~rho~l; r Ls .
The chamois bonding test is a highly appropriate
30 preemptive screening test on a substrate chamois material
derived and processed from viable animal skin with a
matrix collagen protein structure. Processed chemically
into its inanimate state, the chamois retains the same
basic fibrous tissue collagen structure that has been
35 found to be reactive to a variety of bonding systems
2 t 8~228
WOgs/26749 r~ s ~ l987
-- 25 --
described in this and ensuing Examples. Although varying
c~n~ rably across the length and breadth of the stock
material with as much as 10% to 60% standard deviation of
the four averages, the discriminating merit of the
5 chamois test bonding is nonetheless appreciable and
valuable when replications of test specimens are used
with unsparing statistical treatments.
Given the above descriptions of stated superior
productivity, the process ef f iciency and product
10 qualifications now provide for the first time the
collective set of criteria, unknown or unavailable in the
prior art, for the next step of in vivo animal testing of
tis6ue bonding, referred to in surgery as tissue
approximation or as anastomoses, and in turn for approved
15 clinical trials and use.
21 85228
W0 95/26749
-- 26 --
Table 1
Comparison of Prnductivity an- Product Qual f ication
Product A Product B 8/A
Prior Art Example 1
Initial Plasma 40 40 NIA
Volume (ml)
5 C~ enLL~.te 0.515 1.141 3.3/1
yield (gram6)
% solids content 6.24 12.4 2.0/1
dry solids 32 144 4.5/1
content yield
10 (mgms)
Clotted 28 121 4.3/1
f ibrinogen yield
(mgm)
residual 4 23 5.8/1
15 proteins* (mgm~
lapsed time A/B 16.3 2.3 7.1/1
ratio (hours)
relative 26 . 7 83 . 3 3 .1/1
viscosity**
20 (centipoises)
bon~ling strengtb-ch~mois, p~ ~sive throm~in activ~tetl
Tensile break 0.83 5.66 6.811
f orce
( lb-f / in-w)
25 % Regain to 10.1 21.3 2.1/1
control***
% elongation 16.2 33.9 2.1/1
% Regain to 8.7 19.3 2.2/1
control***
30 Calculated, yield .ry solids (c) minus clotte yield
(d) .
**Relative to glycerol standard 1150 centipoises RT,
based on force through clinical syringe 20 G 1 1/2
hypo~rmi r. needle.
35 ***Regain of tensile break force and elongation to that
of the control non-cut chamois stock material.
21 85228
o 9~lZ6749 r.
-- 27 --
EXA~IPLE II
Example II provides a preferred pmho~; r-nt of
fibrinogen concentrates made to at least 30% solids
content by the controlled temperature-time thermal drift
5 process. It also d LLcltes increased productivity
with several stages of repeated cryofreezing of recycled
centrifuged plasma for ~e~uv~:L~ble fractions of
f ibrinogen concentrates comprising the essential, useful
and valuable, symbiotic associated proteins. Also, it
10 ~1 LLc~tes that with the controlled t ~LUL~ time
thermal drift, the separate step of thawing can be
eliminated and carried out simultaneously with the
centrifuging step throughout each stage of the process.
In this example, two recycled supernatants are
15 d ~Lc.ted with substantial increases in the
productivity of the f ibrinogen and associated plasma
glycoproteins as summarized in Table 2, on productivity
and qualification tests far PY~ PPrl;n~ that shown in Table
1.
20 Product - Contrûlled Thermal Drift.
Four aliquots, 38 ml each, of fresh frozen plasma
having initial plasma solids of 9.74%, from extended
storage at -20C (not over three months) containing 0 . 025
ml Kefzol (Lilly) antibiotic solution (1 gm in 2.5 ml
25 sterile water) and 20 mgm of epsilon-~m;nor~rroic acid
(EACA) were placed in sterile 41 ml polypropylene
centrifuge tubes and cryofrozen at -80C for 1 hour.
Without a separate step of thawing, the cryofrozen
aliquots were placed directly in the 4-place Du Pont ~B-4
30 swinging bucket of a Du Pont RC-5C Sorvall Superspeed
Centrifuge. The centrifuge was pre-set to 14C for the
centrifuging time of 32 minutes at 8000 xg. The selected
temperature-time combination with centrifugal force
2 1 85228
Wo 95/26749 r~ 87
-- 28 --
thereby controlled the CU~ ULLe~IL thawing through the
solidus - liquidu6 transition to a residual solid plug of
ice of 15 gm, corr-~-p~n-ling to approximately 40% residual
icing transformed from the crystalline cryofrozen mush
5 during the centrifuged thawing.
The sedimented cryoprecipitates of the f our
aliquots were separated from the decanted supernatant and
ice plug for the first Fraction I. The identical
freezing and centrifuging process steps were repeated on
10 the decanted ~u~e:LIlatant fluids. Table 2 summarizes the
productivity, process efficiency, and qualification
testing for Fraction I and recycled Fractions II and III.
Productivity
This example, as shown in Table 2, accomplishes
15 each of the three intended demonstrations of substantial
increases in productivity of f ibrinogen and its
associated plasma proteins, process efficiency with
elimination of the separate thawing step, and recycling
of supernatants f or ~nhAnrl~cl productivity . It is
20 particularly noteworthy that the three Fractions I, II,
III, the last two of which were ~L uduc~d by recycling the
successive ~u~ Lll~nts starting from Fraction I,
resulted in substantial in.;L ~ Ls of additional
cullc l.LLe,te yields. Starting with Fraction I with an
25 initial .:ul.ce~.LLt.te yield 4. 67 grams (from 4 aliquot
charges of 38 ml of initial plasma totalling 152 ml),
amounting to 32 . 5% of the aggregate of the three
Fractions, the two successive recycled Fractions provided
additionally 36.9% and 31.4% of the valuable fibrinogen
3 0 concentrate with the associated plasma proteins .
Because of the inordinate variability of human
plasma with ; ~Ible components, over some 1000
protein configurations, with a wide range of dry solids
contents, the foregoing productivity and ensuing quality
- - 21 85228
WO9sl26749 1~II~,.,~
-- 29 --
test specif ications can be expected to ref lect
COLL~ in~ variability in yields and quality test
results . The same can be expected f or the inordinate
combinations of the applied variations in temperature,
5 time, and centrifugal in the course of the cryofreezing,
thawing, and centrifugation.
~lotted Eibr; n-~qen AssaY and Yield .
The productivity with the shortened controlled
thermal drift time by the elimination of the thawing step
10 is also evident in the yields of the clotted f ibrinogen
starting from the initial Fraction I with a 1160 mgm
yield, followed successively with the recycled
supernatant Fraction II yield of 1192 mgm and supernatant
Fraction III yield of 428 mgm. As the clotted fibrinogen
15 decreases with each ensuing Fraction, the ULL~ n~7i
yields of the balance of plasma proteins increases.
Thus, as the yield of clotted fibrinogen decreases, the
natively associated proteins increase.
Extrapolation of the recycled productivity under
20 the controlled thermal drift of this Example indicates
that all of the f ibrinogen and associated plasma protein
would be exhausted in the next Fraction IV and Fraction
V . The assay of the clotted f ibrinogen concentrate
content is ~letr-rm;nPd by the ethanol precipitation method
25 described in preceding Example I for each of the three
Fractions reported in Table 2 in terms of percent dry
clotted fibrinogen based on the initial cu~ nLL~te
solids yield expressed in milligrams. The cuLL~ J~ ~linq
I ~ i n~ r of non-clotted product yield constitutes the
30 uLyu~JL~cipitated associated native adhesive
glycoproteins .
Table 2, along with the clotted fibrinogen, shows
the substantial productivity of valuable residual
proteins needed for their adhesive quality. Starting
2 1 85228
Wo 95l26749 1
-- 30 --
with the initial 470 mgm yield in Fraction I,
repre&enting 28 . 8% of the solids yield, the yields
increase progressively with the succ~sive recycled
Fraction II, 1015 mgm, and Fraction III, 762 mgm, to the
5 CuLL~ ;n~ 46.0% and 64.0% composition in the
cryoprecipitated concentrate.
Oualification Tests
Qualification testing is an important feature of
providing specif ic tests related to the intended
10 application to assure expected performance. For tissue
bonding the first and primary requirements include
adequate viscosity for viscous adhesion, similar to that
of glycerine or like a household glue. This imparts
~dequate initial adhesive strength that may be passive or
15 activated by molecular interaction in a short time. The
ensuing tests were devised to provide reasonable
correlation to the expected applications in clinical
tissue bonding.
Viscositv .
Increasing the solids content from 12. 6% as
obtained by Product B of Example I to 34.1% solids in
Fraction I in this Example II signif icantly increases the
relative viscosity from 83 . 3 to 154 centipoises. An
increase in the viscous adhesive quality, particularly
25 important for initial sticking to tissue surfaces, is
thereby provided. Substantial increases in viscosity are
also attained in the sl~cQpp~l;nq Fractions II and III.
The viscosity qualif ication test devised in this Example
is a highly important test used to ascertain the shelf
30 life of stored concentrates in relation to either
increase or decrease of viscosity due to potential
molecular changes involving clotting or fibrinolysis
2 1 85228
~W0 95~26749 P~
-- 31 --
controlled by inclusion of appropriate preservatives and
antibiotics .
Rnn~9;nn strenath. c hAr-is -- ~assive thrnmhin--calillm
svstem.
Table 2 , item (k), summarizes the bonding achieved
by the initial Fraction I followed by the successive
recycled supernatant Fractions II and III. Substantial
regain of tensile bonding strength and elongation,
indicated in percentages compared to that of the control
10 chamois stock is also revealed. The bonding series shown
in Table 2 utilizes 5 to 15 NIH units of thrombin in 5 to
25 microliters of 0 . 5 mM calcium chloride solution,
although with the high concentrate solids the need for
the latter was not evident. The bond strength was tested
15 after 24 hours at room temperature to attain the maximum,
stabilized level of adhesive bond strength, as indicated
by Table 2, Fraction I, of 7.45 lb-f/in-w tensile bond
strength with 32 . 4% regain to the tensile strength of the
stock chamois material. Recycled Fractions II and III
20 with 5.11 and 5.65 lb-f/in-w bond strength and 20.2% and
20 . 59~ regain to control tensile strength regain are
considered acceptable for in vlvo animal testing.
For more rapid tissue bonding, particularly in a
matter of seconds, the passive adhesive bonding can be
25 augmented with thPrr-l ly activated intermolecular
r--h~ni of cross-linking, rl;eAceoriAtion~
polymerization, etc. using spectral p~ LLation or
absorption with endothermic heating as d ~L ~ted in
the ensuing examples.
3 0 Bon~i; n~ strenqth .
Microwave bonrl; n~,
Microwave penetration of the interface between the
fibrinogen cc,~.c~.ll Lc.te and the tissue substrate provided
21 85228
WO 95/26749 P~
-- 32 --
a convenient means for ascertaining the supplemental
contribution of energized molecular motion for
supplementing the passive adhesive bonding. The
preparation of the te6t specimens using the three
S Fraction c.~l- t llLLates applied directly to the joining
edges of the chamois specimen is described in Example I.
The sp~ ir-nc: were then placed in a microwave field o~ a
household unit at a microwave frequency of 2420 ~Iz,
powered by single phase 120 VAC 60 Hz of 900 Watts, at
10 t-~OaUr~ times of 1 to 8 seconds attaining maximum
tensile break strength and elongation usually in 2
seconds, beyond which marked decreases ensue.
The test results shown in Table 2 indicate a bond
strength of 8 . 5 lb-f / in-w in 2 second microwave heating
15 d -LLcLting a substantial increase over=the 6.1 level
attained with the preceding passive thrombin activated
bond strength indicating a thermally induced inter-
molecular bonding augmenting or replacing the passive
bonding. The microwave endothermic bonding served as
20 convenient index frequency energetics from which to
predict caloric absorptions at a broad range of
frequencies to include other frequencies such as ultra-
violet as well as by amplification of stimulated emission
of radiation, for which the su~ce~ in~ example of laser
25 activated bonding of the three Fractions is provided.
The chamois bonding also 6erves as a useful means
for evaluating and comparing the efficacy of fibrinogen
prepared by precipitation with ethanol directly f rom
plasma at room temperature and reconstituted to 36%
3 0 solids in sterile Ringers lactate . In a passive thrombin
activated bonding test, see Table 2, the reconstituted
fibrinogen concentrate attained a tensile break force
averaging 0.31 lb-f/in-w, an unacceptable, risk level for
animal testing, compared to 7.45 lb-f/in-w tensile break
35 force or approximately 1/24 of that attained with the
2t 85228
o gs/26749 1 ~ 1, u ~ _
-- 33 --
cryoprecipitated Fraction I of Table 2. The markedly
inferior bonding attained with the reconstituted high
solids fibrinogen concentrate is attributable to either
the depletion or denaturing, or the combination of both,
5 of the essential residual native adhesive plasma
proteins .
Bondina Strenqth. Chamois - laser activated bondina.
This laser activated bonding uses indocyanine
green dye (Cardio-Green, Becton, Dickinson and Co.,
10 Cockeysville, MD) having a maximum absorption at 805 nm
with an extinction coefficient of 2 x 105 m~lcm~~. Prepared
as a 2% solution, 20 mgm in 1 ml sterile water, it was
admixed with each of the Fraction concentrates at a
proportion of 0 .1 ml of dye solution to 0. 6 ml of the
15 concentrate in a 1 ml 20 G 1 and 1/2 syringe. A bead of
about 1 to 2 mm in diameter of the dyed cG~ ellLLated was
applied in between the butted edges of chamois.
The laser beam was applied from diode laser
module, System 7200 (Spectra Physics, Mountain View, CA)
20 coupled to a hand held focusing optic with a beam
c.r of 2 mm and directed at distance of 4 cm from
the uuil~el~Late bonding applied in 10 timed spots across
the 1/2 inch width of the syringed concentrate. A
progressive series of times per bonding of 10, 20, 40,
25 and 80 seconds was applied in order to determine and
record the optimum time for the maximum tensile bond
strength and elongation and their respective regain to
that of the chamois stock control.
Table 2 , item (m), summarizes the results of the
30 dye absorption laser bonding for the three Fraction
co~ LLates revealing higher bonds strength ~ith
Fraction II compared to Fraction I and a lower bonding
strength with Fraction III. There appears to be a
tlPrPn~lPnry of the bond strength upon some optimum ratio
21 85228
W0 95/26749
-- 34 --
of clotted f ibrinogen to residual proteins, such as
listed in Table 2. However, the range of the ratios with
the three Fractions qualif ies all three ratios f or
effective in vivo animal tissue.
5 In vivo ~nir-l Tissue Testinq.
~ nhAn~P~l fibrinogen cvl,ce,.LL-ltes, 12% to 40% dry
solids content, prepared according to the controlled
thermal drift process. Minimal chamois thrombin
activated bonding strength of 1.2 f~)lc~ puu-lds per inch
lo width, was te6ted along with laser spectral indocyanine
absorption and compared to suturing. This test involved
the rejoining of minimal 5 to 6 cm lengths of dorsal
incisions on Wistar rats, 5 rats for each group of days
of healing, weighing about 450 grams, throughout the
15 healing to the complete restoration of the bi -h;~ni cal
integrity .
The incisions were made under anesthesia in
duplicate on either side of the spine, usually with a
random pairing of different preparations of cull~:llLLc~te
20 pairing cu,.- ~..LL~.tes with sutures, and pairing the three
bonding methods of opposite sides of the spine. The
retrieval ~pec; ~ for tensile rupture force use the
same test 1/2 inch wide strip dimensions as used in the
chamois tests to evaluate the ensuing wound healing
25 during the critical period of 4 to 14 days and the
continuing healing from 14 to 90 days to the complete
tissue restoration. In the case of the thrombin
activated bonding a m;~l;nc;~ion restraint was sutured as
a safeguard against early rampant rupture due to chance
30 hyperactivity. On retrieval of the rejoined incisions at
specified days of healing, appropriate non-incised tissue
strips were taken at each end of the incision as control
n-~n;nc;c:Pd sppi--nc for ~CSPc~;n~ each of the three
means of tissue restoration.
- 21 85228
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-- 35 --
Table 2 summarizes the results of the extended
healing of the f ibrinogen tissue bonding comparing the
passive thrombin activation bonding with that of the
laser spectral absorption and in turn with that of
5 conventional suturing. Based on appropriate statistical
analyses, the three compared bonding modalities are
substantially equivalent with regard to the healed
rupture strength during the critical early healing of 4
to 14 days. During the ensuing tissue healing period of
10 28 to 90 days the thrombin and laser bonding modality are
statistically equivalent, but attain higher rupture
strength than the sutured modality at go days attain the
fully restored, healed bi~ -ch~n; cal integrity.
` ~ 21 85228
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-- 36 --
Table 2
~nhAnced Productivity and Quali- ication Tests
Processed I II* III* Aggregate
Fractions
5 Initial 38 x 4 36 x 4 34 x 4 152
Plasma
Volume (ml)
concentrate 4 . 67 5 . 98 3 . 79 14 . 48
yield
10 ( grams ~
% solids 34.9 36.9 31.4 N/A
content
Solids 1630 2207 1190 5027
yield, dry
15 (mgm)
clotted 1160 1192 428 2780
fibrinogen 71.2 54.0 36.0 N/A
(mgm) %
residual 470 1015 762 2247
20 protein 28 . 8 46 . 0 64 . 0 N/A
(mgm) %
fibrinogen/ 2.47/1 1.17/1 0.56/1 N/A
protein
ratio
25 lapsed time 1. 5 3 .1 4 . 6 N/A
( hours )
Relative 154 131 125 N/A
viscosity **
( centi-
30 poises)
Bon~ing ~tr~ngth - Chamois, P~ssive Thrombin
Activat~ R~r, 2 4 ~ 8 .
tensile 7 . 45 5 .11 5 . 65 N/A
break f orce
35 lb-f/in-w
% regain to 32.4 22.2 20.5 N/A
control***
% Tensile 27 21 20 N/A
elongation
40 % regain to 41 29 31 N/A
control***
2 t 85228
W0 95126749 ~ J 'h _
-- 37 --
Proce6sed ¦ I ¦ II* ¦ III* ¦ Aggregate
Fractions
Bon~ing -Itrength - c~amois, mic~owave 2 secon~
Tensile 8 . 5 9 . 54 8 .18 N/A
break f orce
lb-f / in-w
5 % regain to 32.3 36.2 31.1 N/A
control***
% Tensile 19.8 20.8 19.3 N/A
elongation
% regain to 38.2 40.1 37.3 N/A
control***
Bonding strength - chamois, lase dy~ ~bsorption
Tensile 2 . 62 1. 73 1.14 N/A
break f orce
lb-f/in-w
% regain to 10 .1 6 . 56 4 . 33 N/A
control***
% tensile 18 . 0 17 . 6 16 . 0 N/A
elongation
% regain to 34.6 33.8 30.8 N/A
control***
In Vivo Rat incision - healing, tensile break force
( lb-~/ in-w)
ret'l days 4 7 14 28 60 90
suture 0 . 58 2 . 2 4 .1 15 . 5 48 . 4 48 . 7
ref.
****% 0.74 2.6 5.3 19.1 53.5 62.5
regain
thrombin 0.53 2.7 5.3 18.5 52.0 81.3
activated
****96 0.69 3.1 6.8 22.9 57.4 104.4
regain
laser dye 1.1 3.1 5.7 19.6 53.2 76.1
absorption
****% 1.4 3.8 7.2 24.3 58.8 97.6
3 5 regain
. .
21 85228
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-- 38 --
* Recycled supernatant from preceding processing stage.
** Relative to glycerol standard, 1150 centipoises,
forced through l ini~l syringe, 20 G 1 1~2 hypodermic
needle .
5 *** Regain of tensile break force and elongation
compared to control, non-cut chamois 6tock material.
**** Regain of tensile break force compared to that of
control, non-cut adjacent dorsal skin.
ret ' l = retrieva l
10 ref. = reference
-
21 85228
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-- 39 --
EXAMPLE I I I
The purpose of this example is to prepare
cryoprecipitated fibrinogen concentrates from a series of
mixtures of pooled plasma with added albumin. Albumin
5 was included to supplement f ibrinogen concentrates with
the adhesive glycoproteins and the low molecular weight
pre-~lhuminc that contain valuable factors for cell
growth for healing of incised tissues bonded with the
fibrinogen CUllCt~ L~teS. It is evident from the
10 preceding Example II that recycling cryoprecipitated
fibrinogen concentrates, as shown in Table 2, resulted in
surprising increasing yields and increasing proportions
of the associated residual native plasma proteins, i.e.,
inverse fibrinogen to plasma ratios, and provided useful
15 and effective levels of adhesive tissue bonding as well
as substantial productivity of a clinical product.
Pr~n~ration of Concentrates
The controlled thermal drift process described in
Example II was applied to a pIuyLaSsive series of human
20 plasma - human albumin compositions using unused portions
of collected pooled plasma of varying refrigerated
storage times up to 11 months at -20C. The pooled
plasma was supplemented with clinical, U. 5 . P . grade
albumin (human 25 solution) in a ~LuyL~ssive series of
25 proportionate levels of o%, 10%, 20%, and 40% admixture.
Table 3 summarizes the principal specif ication items
wherein the albumin supplemented not only the
productivity of the process but also replaced a
significant portion of the plasma for the qualification
30 tests for adhesive bonding. The plasma was cryofrozen at
-80OC for about one hour. Thawing and centrifugation
were performed simultaneously at 14C for 32 minutes at
8000 xg.
- 21 85228
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-- 40 --
ProductivitY
The admixture with albumin sustained the solids 35
content above 30%, see Table 3, starting from the 31.5%
level with the control non-admixed plasma followed by a
5 marked increase in the solids yield, also see Table 3,
over that of the control. This indicated a significant
and 6urprising yield of clotted fibrinogen along with
associated native proteins for their adhesive value.
Thus, in a single production stage, without recycling the
10 supernatants, the one single uLyuuL~cipitated Fraction I
of the admixtures with albumin is a novel and highly
useful product for adhesive bonding. It is noteworthy,
nPYpectefl and surprising, that the supplementation with
albumin induces marked molecularly associated
15 cryofreezing with substantial solids concentration and
yields of clotted f ibrinogen across the entire range of
albumin admixtures.
Compared to Product A of Example I, typical of the
prior art with a yield of 28 mgm per 40 ml volume of
20 plasma, the 60/40 plasma-albumin composition yields 280
mgm, see Table 3, amounting to 10 times the amount of
clotted fibrinogen from Product A. One knowledgeable
about the protein components of albumin would not expect
the inordinate level of recovering clotted f ibrinogen .
25 gualif ication Tests
The admixtures of the human plasma with human
albumin resulted in signif icant and consistent increases
in the relative viscosity, shown in Table 3, thereby
providing Pnh~ncPcl viscous contact, a quality of
30 stickiness, to tissue substrates. In the chamois passive
adhesion bonding strength, Table 1, using thrombin-
calcium chloride activation, the albumin supplementation
provided significant Pnh~n~ -nt in tensile break force
and elongation commencing at 20% to 40% level. Similar
21 85228
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-- 41 --
~nh2~r- t was made evident in the chamois adhesive
bonding by means of spectral microwave, and by laser dye-
absorption, bonding commencing at the 10% to 40% level of
albumin supplementation As stated in the preceding
5 Example II, considerable variations in the extent of the
attained ten6ile break strength was made evident with the
three different bonding systems each having optimal
maximal ~nh~n~ -nts of tensile break strength and
elongation d~r~n~in~ upon applied thermal or spectral
10 energy and the pertaining time of absorption. In the
series of this example, the microwave activated bonding
is a superior and easier means of tissue bonding over the
other two.
21 85228
Wo 95lZ6749 1 ., I / 1, ., _. . _
-- 42 --
Table 3
Productivity and Qualification Tests
Suppleme~ltation of Plasma wit.h Albumin
Plasma Albumin 100/0 90/10 80/20 60/40
5(vol/vol)
plasma (ml) 38 36 . 2 30- 4 22 . 8
albumin (ml) 0 3 . 8 7 . 6 15 . 2
Proauc ivity rec,~cle~ fr~c-ion series
Concentrate 1.333 1.614 1.845 1.762
10 yield (grams)
% ~:olids 31.5 34.8 34.0 36.3
content
dry solids 420 562 627 640
yield (mgm)
15 Clotted
f ibrinogen
mgm 354 411 407 280
% 84.3 73.1 64.9 43.8
residual
20 proteins*
mgm 66 151 220 360
% 15.7 26.9 35.1 56.2
fibrinoqen 5.36/1 2.72/1 1.85/1 1/0178
protein ratio
25 Viscosity** 157 170 173 168
centipoises
Bonding 8trengt - Passive ,",~ ~ ;n Activ~ted ~T 24 hrs
tensile break 1.20 1.20 1.80 3.20
lb-f / in-w
30 % regain to 4.6 4.6 6.8 12.2
control***
% Tensile 7.6 8.1 10.8 16.4
elongation
% regain to 12 .1 13 . 7 18 . 3 17 . 7
35 control***
Bonding Strength - microwave 2 seconds
tensile break 2.23 3.54 3.70 5.12
lb-f / in-w
% regain to 7 . 8 12 . 8 12 . 8 17 . 9
40 control***
. .
: ` 2 1 85228
WO 95126749 P.
-- 43 --
% Tensile 18.9 20.0 24.7 33.2
elongation
% regain to 36.5 42.0 52.4 64.0
control ***
Bonding 8trength - La er Dye Ab lorption 10 8ec
tensile break 1.45 2.40 2.30 3.33
lb-f / in-w
96 regain to 5.0 8.3 8.0 11.6
control***
10 % Tensile 12 . 3 13 . 0 17 . 8 18 . 8
elongation
% regain to 23 . 7 27 . 3 39 . 2 55 . 5
control ***
*Calculated, dry solids yield tc) minus clotted
15 fibrinogen (d).
**Relative to glycerol standard, 1150 centipoises RT,
based on force through clinical syringe, 20 gauge 1 1/2
hypodermic needle.
***Regain of tensile break force and elongation to that
20 of the control non-cut chamois 6tock material.
21 85228
Wo 95tZ6749 I
-- 44 --
EXAMPLE IV
The purpose of this example is to extend the
albumin plasma supplementation with recycled - u~eL~atllnts
throughout the repeat stage of controlled thermal drift
5 from cryofreezing to centrifugation. This example also
.1 LLcltes increased productivity of the recycled
Fraction concentrates for a wide range of useful
f ibrinogen/protein ratios .
Prenaration of Recvcled Concentrate Fractions
The controlled thermal drift process described in
Example II was applied to a progressive series of
recycled supernatants of single donor human plasma
supplemented with the initial admixture of 40% (vol/vol)
human albumin, 25 U.S.P. grade, into five cu..se.;uLive
15 repeat stages. Table 4 summarizes the resulting
principal specif ications of the items wherein the albumin
supplements needed productivity, but also replaces a
significant portion of the valuable human plasma, as in
situations of limited single donor availability such as
20 pediatric and elderly cases. The plasma was cryofrozen
at -80C for about one hour. Thawing and centrifugation
were performed simult~n~ol~cly at 14C for 32 minutes at
8000 xg.
ProductivitY
The admixture of 40 parts albumin to 60 parts of
single donor plasma lot provided about 3 0% higher yield
of clotted fibrinogen, Table 4, than that of the same
admixture using pooled human plasma in the preceding
Example III, initial Fraction I. This was expected from
30 the variations in the quality of human plasma which is
inordinately variable and ever changing chemically and in
molecular configurations on even few days or hours of ex
21 85228
Wo 95l26749 ~ ).,,5/ ~987
-- 45 --
vivo storage, notably with f ibrinolysis and
intermolecular associations, In this example, listing
only the Fraction I productivity, the fibrinogen/protein
ratio, 6howed approximately 75% higher proportion of
5 clotted f ibrinogen . These specif ications of the
productivity invoked substantial differences in useful
quality as ~let ~ cl in the ensuing section and further
emphasized the inadequacy of the prior art in
anticipating or predicting useful qualities.
10 Oualif ication Test6
The admixture with 40 parts of albumin provided a
modest level of viscosity, substantially Pnh Inr.P~ with
successive recycling throughout all four Fractions
attaining a maximum at Fraction III. This provided an
15 important feature of performance in surgical applications
for sticking or adhering to tissues during surgical
applications. It is particularly surprising and
unexpected from known prior art that such progressive
~r~cPc in clotted fibrinogen to as low as 3.4%, provide
20 substantial bonding strengths throughout the entire
recycled Fraction series, see Table 4. It may be
~Yrect~cl that in tissue adhesion or bonding each Fraction
upon further in vivo trials in living tissues can be
expected to favor some one particular fibrinogen/protein
25 ratio not only in instant or immediate but also on
prolonged healing to complete bi --h~n;cal restoration
in specific terms of regained tensile break ~Lr~ and
elongation .
21 85228
WO 95126749 . ~ 987
-- 46 --
Table 4
Productivity Ind Quali ication Tests
FRACTION
Albumin I II III IV V
5 (vol/vol~
plasma (ml)
albumin (ml) 22.8 N/A N/A N/A N/A
15 . 2 N/A N/A N/A N/A
Cu~lcel~Llc~te 1.66 1.49 2.38 1.39 N/A
yield (grams)
10 % solids 34.3 38.5 41.5 39.7 N/A
content
Clotted
Fibrinogen
(mgm) 363 240 24.0 19.5 647
15 % 63.6 41.7 2.4 3.5 N/A
residual
proteins*
mgm 208 336 963 532 2038
% 36.4 58.3 97.6 96.5 N/A
20 fibrinoqen 1.75/1 1/0.72 1/40 1/27 N/A
protein ratio
Viscosity** 91 109 204 145 N/A
centipoises
Bonding strength - chamois, Passive thrombin
activ~t~d R~, 24 hrs
Tensile break 6.13 6.30 3.50 4.47 N/A
lb-f / in-w
96 regain to 21.3 24.5 12.1 14.6 N/A
control***
30 % tensile 13.3 12.0 8.0 7.0 N/A
elongation
% regain to 25.5 23.1 15.0 13.5 N/A
control ***
Bonding strength - chamo .s microwave 4 s~c
35 Tensile break 5 . 68 4 . 35 5 . 45 4 . 90 N/A
lb-f / in-w
% regain to 15 . 9 12 .1 15 . 2 13 . 9 N/A
control ***
% tensile 16 . 8 27 . 3 33 . 0 12 . 0 N/A
40 elongation
.... . _ _ . . ..
21 85228
Wo 9s/26749 F~
-- 47 --
% regain to ¦ 32.3 ¦ 52.5 ¦ N/A l N/A ¦ N/A
control** *
Bonding ~-rengtb - Ch~mois l~ser dye ~bsorpti-~n
Tensile break 4 . 3 4 . 0 4 .1 3 . 6 N/A
5 lb-f / in-w
% regain to 12 .1 11. 0 11. 5 10. 0 N/A
control** *
% tensile 14 .1 18 . 0 14 . 2 8 . 6 N/A
elongation
10 % regain to 21.5 27.5 21.7 13.1 N/A
control***
*Calculated, dry solids yield (c) minus clotted
f ibrinogen (d) .
**Relative to glycerol standard, 1150 centipoises RT,
15 based on force through clinical syringe, 20 c, 1 1/2
hypodermic needle.
***Regain of tensile break force and elongation compared
to that of control, non-cut chamois stock material.
21 85228
WO 9S/26749 1 ~ ~ J87
-- 48 --
EXaMPLE V
This Example d ~ LLtltes the productivity and
q~lAlificAtiong of bovine fibrinogen concentrates product
as the extension of the controlled thermal drift process
5 to other r l; An plasma.
Pre~aration of Concentrates
The same procedures from cryofreezing to
centrifugation as described in Example II were applied to
bovine plasma. The leading Fraction I serYes to
10 establish the process efficiency of the selected
temperature-time conditions, and at least two recycled
Fractions T for gaining substantial proportions of the
Associated native proteins and particularly the plasma
bioadhesives. Table 5 summarizes the principal
15 specif ications of productivity and qualif ication tests
using a commercial source of bovine plasma with an
initial plasma solids assay of 14 . 3% . This example
illustrated the general applicAhil ~ty of the temperature-
time thermal drift process and the resulting PnhAnrPr~
20 fibrinogen concentrate products from different variants
of ulyu~Lecipitated types of viable plasma. The plasma
was cryofrozen at -80C for about one hour. Thawing and
centrifugation were performed simultaneously at 14C for
32 minutes at 8000 xg.
25 ProductiYi~Y
It is noteworthy that the leading initial Fraction
I from bovine plasma provided substantially higher
concentrate yields more than 2 times (2.75) that of the
average (1.33) attained in the preceding Examples with
30 human plasma, and even higher with the successive
recycled Fractions II and III. The successive series o~
Fractions d -- LLclte the consistent general trend of
increasing clotted f ibrinogen yiel~s along with that of
_ _ _ _ _ _ ,, , _ , , _ .. _ ....... _ .. . .
21 8522~
w0 95l26749 r~ l~u~,_lP~s87
-- 49 --
the residual proteins as in the case with the human
fibrinogen shown in Example II, Table 2, with pronounced
effects on the ensuing qualification tests.
011 11 i f ication Tests
The leading qualification of viscosity increased
substantially with the successive recycled Fraction
series imposing a pr-mrnlnr-~d effect of the bonding
quality. In the case of passive thrombin activated
bonding the increase continued consistently from Fraction
10 I to Fraction III. In the case of the microwave
th~ l ly activated bonding which was most effective of
the three sets of bonding, the same consistent increase
from Fraction I to Fraction III prevailed. The bonding
strength in the case of the laser dye-absorption also
15 resulted in a pronounced increase from Fraction I to
Fraction II followed by a pronounced decrease with
Fraction III. This increase appears to be due to either
excessive laser .:~oauL~ time or a depletion of a
bioadhesive protein component but with a useable and
20 effective level of bonding equivalent to that of Fraction
I.
In this chamois bonding series, a reconstituted
36% cu.lcel.LL~te of bovine fibrinogen, Type I-S of a
commercial source, in Ringers lactate was bonded by
25 microwave ~X~JOaUL~ for 2 seconds, see Table 5. The
resulting tensile break force of 0. ~2 lb-f/in-w was
significantly inferior to 7.63 lb-f/in-w break force, or
about 1/8 of that attained by bovine Fraction I of this
example. This indicated that the reconstituted source of
30 bovine was lacking in both the intrinsic adhesive quality
and the r ,.l.u--se to thermal bonding incurred during the
processing, presumably denaturation, from the native
aqueous Cu...:e-,l Lat~ state to the dried dehydrated powdery
form .
2 1 85228
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-- 50 --
In Vivo Animal Tissue Te5tina.
The same dorsal inci6ion bonding on Wistar rats as
described in Example II was applied to the time-extended
fieries of retrievals in the qualification tests for
5 clinical applications. Table 4 summarizes the results of
the retrieved laser dye absorption bonded test specimens
comparing the tensile break or rupture force pairing the
human and bovine f ibrinogen cu~lct:i-LL ~te on opposite
dorsal sides of incisions f or the initial critical period
10 of 4 to 28 days of healing. The results indicate that
the bovine and human f ibrinogen concentrates were
substantially equivalent in developing gradually the same
rate of healing in terms of the attained tensile break or
rupture force and the proportionate regain in 28 days to
15 that of the control non-incised tissue.
2 1 8522~
W095/26749 r~
-- 51 --
Table 5
Productivity and Qualification ~ests
of Bovine Fibrinogen Concentrates
Fractions I II III Aggregate
5 Concentrate 2 . 75 4 . 78 5 . 57 13 .1
yields
(grams)
~ solids 38.3 37.5 37.4 N/A
content
10 solids 1052 1789 2084 4925
yield dry
(mgm)
clotted
f ibrinogen
15 mgm 912 1274 773 2959
% 86.7 71.2 37.1 N/A
residual
proteins*
mgm 140 515 1311 1966
20 % 13.3 28.8 62.9 N/A
fibrinoqe" 6.51/1 2.47/1 0.59/1 N/A
protein
ratio
viscosity 162 270 426 N/A
25 **
centipoises
Bon~ing Strongth - chamois thrombin ~Lctivnt~l
RT 2 4 hrs
tensile o . 87 1. 20 2 . 30 N/A
3 0 break f orce
lb-f / in-w
9~ regain to 2 .1 2 . 9 5 . 5 N/A
control***
% 2.8 3.6 4.0 N/A
35 elongation
% regain to 3 . 4 4 . 3 4 . 8 N/A
control***
21 85228
W0 95l26749 r~
-- 52 --
Elonding 8trength microw~ve 2 sec
tensile 7 . 63 6 . 4& 6 . 68 N/A
break f orce
lb-f / in-w
5 % regain to 31. 0 26 . 3 29 .1 N/A
control***
% 18 . 7 14 . 7 15 . 3 N/A
elongation
% regain to 19 . 4 15 . 3 15 . 9 N/A
10 control***
Bonding 8trength - chamois laser dy~ ~bsorption
10 sec
tensile 3 . 55 6 .15 3 . 80 N/A
break f orce
15 lb-f / in-w
% regain to 14 . 4 24 . 9 15 . 4 N/A
control***
% 45 . 2 47 . 3 47 . 2 N/A
elongation
20 % regain to 47 . 0 49 .1 49 . 0 N/A
control***
*Calculated, yield dry solids (c) minus clotted yield
(d) .
**Relative to glycerol standard, 1150 centipoises RT,
25 based on force through rl;n;c~l syringe, 50 G 1 1/2
hypodermic needle.
***Regain of tensile break force and elongation to that
of the control non-cut chamois stock material.
21 85228
WO95/26749
-- 53 --
Modif icatiDns and E~l] i valents
The herein described Examples of preferred
;r-nt, cryofreezing, thawing, and centrifuging, to
produce ~nh~n--P~ vi ~coA~lhP~ive fibrinogen concentrates
5 may be further modified with adjustments in the
controlling interactions of temperature x time x
centrifuging gravitational force (xg) other than that
described in the preferred: ` ~ir-nt Example II. For
example, thawing and centrifuging may take place
10 simultaneously. Such process modifications for adjusting
the productivity, process efficiency, and qualification
test specif ications are described in the Application
Serial No. 07/562,839. Modifications produce Pnh~nrP~
~;Ly-J~L 'Cipitated concentrates from about 12% to as high
15 as 40% solids of useful and effective v; cc~ hPC;ve
concentrates for tissue bonding. This high solids range
has been achieved by limiting the thawing at the solidus
- liquidus transition to at least 30% and less than about
95% residual icing. This prevents or minimizes the re-
20 dissolving of cryoprecipitated plasma proteins into theliquidus state. Example II in this application was
controlled to within the 30% to 95% range with 40%
residual icing with implied option of increased or
decreased de-icing as a means f or modifying the native
25 fibrinogen native plasma proteins ratio. It is also
shown in Example II that the process of uses the
simultaneous thawing and centrifuging as a single step of
the process.
Moreover, another salient modification shown in
30 the Examples is the ~roy, ~s~ive recycling of the spent
supernatants to yield Fraction series of concentrates
with an assay of pLoyLessive lowering of the
fibrinogen/residual protein ratios but effective in ex
vivo tissue adhesion. The Fraction series can be used to
21 85228
WO 9S/26749 ~ 987
-- 54 --
make compo6ite admixtures to stated product
specification6 adjusted for solids content and/or the
fibrinogen/native protein ratios where appropriate in
specif ic types of ti66ue bonding or re6tructuring.
The foregoing di6closures and descriptions of the
qualification tests for, and accomplishing viscous
adhesion and passive and/or spectral absorptive bonding
may be ~ Liately modified to the degree of desired
bonding strength. The latter would apply to some
10 preferred minimal solids concentration standard between
12% and 40% or more h:~nrll ;n~ in surgical application
rliRpl~nl:Pr~ from syringe at a preferred range of visc06ity.
It may, by per60nal choice be other than the mid range
nominal 36% 601id6 u6ed in Example II, either higher or
15 lower. Thi6 al60 applie6 to the varying choice of the
optimal fibrinogen/re6idual protein ratio ~erc~n~l;nlJ upon
the type of the anatomical ti66ue, for in6tance, from
exterior 6kin 6tructure to f ine internal va6cular or
gastrointestinal to relatively thin, often of microscopic
20 dimensions and delicate ophthalmic and neural sheath
tissues. In this wide range of tissue 6tructures, it is
expected that each of these types may require a different
set of specifications for optimal, from low to high
solids content and likewise clottable fibrinogen/residual
25 protein ratios for the desired viscosity and tissue
adherence of bonding.
The products of this invention may also be used to
coat woven or knitted graft prosthesis to contain
internal hemorrhaging, fluid seepage, and the like, and
30 to replace or augment suturing as a means of recl--~in~
sutured rigidity. The products of the present invention
are useful in a wide range of surgical tissue bonding,
joining, or restructuring applications by various
techniques such as passive thrombin-calcium activation
2 1 85228
Wo95/26749 r~ 0~987
-- 55 --
involving fibrinogen polymerization and spectral
absorption with directed laser.
Various modif ications of the invention in addition
to those shown and described herein will be apparent to
5 those skilled in the art from the foregoing description.
Such ~odifications are also intended to fall within the
scope of the appended claims.
