CA2185228A1 - Cryoprecipitated native fibrinogen concentrates - Google Patents

Abstract

The present invention is directed to a cryoprecipitated fibrinogen concentrate of native mammalian 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 about 8 lb-f/in-w.

Description

~WO 95126749 2 ~ 8 5 2 2 8 F~ U.. _. . /

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 Wo 9S/26749 PCT/US9~/03987 1--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, Wo 95/26749 P~ I I u,. _ _ 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 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 wo 95l26749 .

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 W0 9~/26749 .

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.

~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.

WO 95/26749 I ~IIIJ~ ~987 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 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) WO 95/26749 I ~ ~

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 .

,. ~
~WO 9C126749 P~l/u.,,~,r~'t87 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 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 ~¦IWO gs/26749 P~~ 987 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 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~ , 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 WO 95l26749 .

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.

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 WO 95l26749 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 WO 95~267q9 1 ~ . IQ~987 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 W09S/26749 1~I,~J.,,~ _, 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~

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, Wo ss/26749 1 . 1, ., . _ , .

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 Wo 95/26749 1 ~l,I 'A~987 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 -Wo gs/26749 ~ 987 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 Wo gs/26749 E~

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 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.

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.

o 9~lZ6749 r.

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 Wo 95/26749 r~ 87 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 WO9sl26749 1~II~,.,~

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 Wo 95l26749 1 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 ~W0 95~26749 P~

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 WO 95/26749 P~

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 ~ _ 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 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.

WO 95l26749 PCr/UssSI039~7 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 W0 95/26749 p~

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 _ 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 . .

W0 95l26749 r~ g87 * 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 -Wo 95/26749 ' ~ " " '3' ~

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.

W0 95/26749 .

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 WO 95/26749 r~,l~J.,,.".

~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.

Wo 95lZ6749 1 ., I / 1, ., _. . _ 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.

% 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.

Wo 95tZ6749 I

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 Wo 95l26749 ~ ).,,5/ ~987 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 .

WO 95126749 . ~ 987 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 .... . _ _ . . ..

Wo 9s/26749 F~

% 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.

WO 9S/26749 1 ~ ~ J87 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 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 .

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~

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***

W0 95l26749 r~

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.

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 WO 9S/26749 ~ 987 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 Wo95/26749 r~ 0~987 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.

Claims (19)

1. What is claimed is:
   l. A cryoprecipitated fibrinogen concentrate of native mammalian 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 450 centipoises, and a tensile break force of about 1 to about 8 lb-f/in-w.
2. 2. The concentrate of claim 1 wherein said plasma is human plasma comprising about 12% solids content, wherein about 84% is clottable fibrinogen, having a viscosity of from about 80 to about 85 centipoises, and a tensile break force of about 5 to about 6 lb-f/in-w.
3. 3. The concentrate of claim 1 wherein said plasma is human plasma comprising about 30% to about 40% solids contents, wherein about 35% to about 75% is clottable fibrinogen, having a viscosity of from about 120 to about 160 centipoises, and a tensile break force of about 5 to about 8 lb-f/in-w.
4. 4. A concentrate of native human 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 450 centipoises, and a tensile break force of about 18 to about 19 lb-f/in-w at 28 days, about 51 to about 53 lb-f/in-w at 60 days, and about 80 to about 83 lb-f/in-w at 90 days post in vivo incision healing.
5. 5. The concentrate of claim 1 wherein said plasma is human plasma and further comprising human albumin, said cryoprecipitate comprising about 30% to about 45%
   solids content, wherein about 40% to about 90% is clottable fibrinogen, having a viscosity of from about 90 to about 175 centipoises, and a tensile break force of about 1 to about 7 lb-f/in-w.
6. 6. The concentrate of claim 5 wherein said plasma and said albumin are combined in a ratio selected from the group consisting of 100:0, 90:10, 80:20, and 60:40.
7. 7. The concentrate of claim 1 wherein said plasma is bovine plasma comprising about 35% to about 40% solids content, wherein about 35% to about 90% is clottable fibrinogen, having a viscosity of from about 160 to about 430 centipoises, and a tensile break force of about 1 to about 2.5 lb-f/in-w.
8. 8. The concentrate of claim 1 wherein said mammalian plasma is selected from the group consisting of human, bovine, porcine, rabbit, and equine plasma.
9. 9. The concentrate of claim 1 comprising about 12% solids content.
10. 10. The concentrate of claim 1 comprising about 24% solids content.
11. 11. The concentrate of claim 1 further comprising albumin.
12. 12. The concentrate of claim 1 further comprising components selected from the group consisting of Factor XIII, mucoproteins, glycoproteins, fibronectin, plasminogen, prothrombin, thrombin, transferrin, and cell growth factors.
13. 13. The concentrate of claim 1 further comprising components selected from the group consisting of anticoagulants, antifibrinolytics, coagulating agents, viscosity modifiers, bioadhesives, surfactants, antibiotics, and preservatives.
14. 14. A tissue adhesive comprising a cryoprecipitated fibrinogen concentrate of mammalian 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 about 8 lb-f/in-w.
15. 15. The tissue adhesive of claim 8 wherein said mammalian plasma is selected from the group consisting of human, bovine, porcine, rabbit, and equine plasma.
16. 16. The tissue adhesive of claim 8 comprising about 12% of solids content.
17. 17. The tissue adhesive of claim 8 comprising bout 24% solids content.
18. 18. The tissue adhesive of claim 8 further comprising albumin.
19. 19. The tissue adhesive of claim 8 further comprising components selected from the group consisting of anticoagulants, antifibrinolytics, coagulating agents, viscosity modifiers, bioadhesives, surfactants, antibiotics, and preservatives.