CRYOPRECIPITATED NATIVE FIBRINOGEN CONCENTRATES

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 1b-f/in-w.

Description: TITLE OF THE INVENTION CRYOPRECIPITATED NATIVE FIBRINOGEN CONCENTRATES Background of the Invention Fibrinogen is one of the numerous proteins of blood plasma from which the phenomenon and mechanism emanate to form the structure of fibrin clot. Its ubiquitous physiological role in internal restructuring or repair of tissue discontinuity has been extended to corresponding role of external application developed over the past scores of years as a concentrate processed from plasma for tissue bonding under such descriptive terms as fibrin clot, fibrin adhesive, fibrin weld, fibrin sealant, tissue sealant, and so on. The clinical use of fibrin prepared from plasma by various methods of precipitation and chemical insolubilization has gradually emerged for such early clinical uses as hemostyptic adhesive powder with small opening vessels (Bergel, S., Deutsch . lied . 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. et al., Wien . tied . Wochenschr. , 1952, 122:517-591), and gradually expanding to the repair of traumatized tissue (Brands, . et al., World J. Surg. , 1982, 6:366-368), and the anastomoses or restructuring of cardiovascular or severed cardiovascular, colon, bronchial, nerve endings 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 clinical assays, ranging from 229 to 742 mgm/dcl standard deviation, based on the photometric measurements of turbidity from clotting (Castillo, J.R. , et al., Thrombosis , 1989, 55:213-219). This range of concentration corresponds to 0.229% to 0.742% (average of 0.513). In a typical prior art example such as Dresdale et al., Surgery, 1985, 97:750-754, the time sequence of cryofreezing, thawing, and centrifuging produces a fibrinogen concentrate of only 2.16% (2160 mgm/dcl). The resulting fibrinogen concentrates typical of the prior 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 requires standby chilling resulting in lack of viscous adhesiveness needed for manageable and effective surgical 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 product qualifications with supporting tests for manageability, viscosity, adhesion, and effective high solids fibrinogen concentrates for surgical reconstitution of severed or incised tissue. One conventional means for separating or concentrating fibrinogen from mammalian plasma is by various chemical precipitating procedures of admixtures with concentrated salt solutions, such as semi-saturated sodium chloride, saturated ammonium sulfate, and by cold ethanol and other low molecular weight organic compounds, notably amino acids such as glycine, and numerous combinations thereof. These chemical precipitating procedures imposing varying degrees of denaturization in contrast to the non-chemical cryoprecipitation methods which are applied to the preparation of high purity, single fibrinogen entities stripped of the nascent, natively associated symbiotic plasma proteins which may remain dissolved in the added chemical precipitants. The nascent, hereinafter termed native, proteins include glycoproteins of various configurations with carbohydrate 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 significant adhesive tensile strength in the measurements of the bonding strength described in the various examples in the present invention. A cryoprecipitate of native fibrinogen heretofore has not been generally recognized as a preferred source of enhanced high solids fibrinogen concentrates. 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 chemical stripping imposes 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 performance. Cryoprecipitation imposes structural changes in the plasma proteins due to fibrinolysis during the prolonged cryogenic state of conventional methods in terms of the inevitable temperature-time kinetics. The prior art has disclosed the use of a wide range of temperature-time variables but provides no indication of the effect of varied temperature-time kinetics on productivity and product quality on the first procedural step of cryofreezing. Rather, the prior art literature infers that longer periods of cryofreezing and thawing is necessary for attaining higher purity of the fibrinogen. For instance, the clinical preparation commences by cryofreezing at -80°C specified for at least 6 hours (Gestring, G.F., et al., 1982) later this was increased 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 clinical preparations provides data on productivity and qualifications of the resulting cryoprecipitated products in support of the need for increasing the stated cryofreezing 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 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 cryofreezing. Apart from reflecting a substantial increase in process efficiency, this reduction in cryofreezing time makes the process and product suitable for urgent need of autologous clinical preparations. Obviously, for immediate autologous clinical usage for ready surgical availability only one hour of the prolonged cryofreezing times is proven to be highly acceptable. In none of the prior art descriptions has there been any consistent indication of both the concentrate yield and the product characterization and quality testing. Following the cryoprecipitation process step, thawing is the next essential component of the process during which the solid heterogeneous crystalline-like frozen mush is transformed into two phases of sedimented precipitate and a viscous fluid with a glacialized homogeneous solid plug of ice, hitherto not recognized in known prior art. With prolonged thawing, either as the usual separate step or simultaneously during centrifugation, the ice progressively 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 the resulting concentrate yield, the solids concentration, and the distribution of the numerous associated plasma proteins through the solidus - liquidus equilibrium transition temperature depicted as follows: Process phases cryoprecipitation > thawing > centrifugation 0°C (solidus) (de-icing) (liquidus) wherein the frozen solid plasma releases the cryoprecipitated insoluble fibrinogen and relatively soluble associated proteins which are important for the fibrinogen concentrates in tissue bonding and controlled to desired contents in the concentrates. The ratio of the fibrinogen associated proteins thus can be regulated by the thermal drift of the solidus to liquidus transition 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 to the fibrinogen and also intended to be retained as much as possible by the temperature-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 at temperature for any required retention for the adhesive quality in tissue bonding. The retention of the associated plasma proteins is highly dependent upon the thermal drift from the cryogenic state through the icing equilibrium with minimal time in the liquidus watery phase during which the associated proteins begin and continue to re-dissolve from the cryoprecipitated state. The thawing time in numerous known, published methods is not consistent and in no instances correlative to either the quantity or quality of the attained fibrinogen concentrates. For instance, the specified thawing time varies from such indefinite temperature-time kinetics as at 4°C "when liquid" (Gestring, supra) ; at 4°C "for several hours" (Dresdale, supra) ; and at 1°C to 6°C 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- dissolving of the cryoprecipitates during the temperature-time thermal drift with inordinate loss of fibrinogen and its associated proteins with solids contents ranging from as little as 3% to 6%. The need for minimizing the temperature-time thermal drift was made evident in Application Serial No. 07/562,839, by which the products of this invention have been produced. Following thawing, the cryoprecipitate is subjected to physical separation by centrifuging at specified gravitational (xg) force in the course of the temperature-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, unspecified cold centrifuge at 2300 xg for 10 minutes to 15 minutes (Gestring, supra) , 1000 xg for 15 minutes (Dresdale, supra) , 5,000 rp (unspecified xg) at 1°C to 6°C for 5 minutes (Sidentop, K. H. , supra) , and at 6500 xg for 5 minutes at 4°C (Spotnitz, supra) , again with no indicated productivity and qualification tests. 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 criteria of quality for safe, effective and reliable uses in surgical tissue bonding to which the products of this invention are directed. Summary of the Invention 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 81b-f/in-w. The invention is directed to a cryoprecipitated native, undenatured, non-lyophilized fibrinogen concentrate. The fibrinogen concentrate of the present invention may be associated with nascent indigenous proteins which enhance the viscous adhesion in tissue bonding. Another objective is to provide a high solids fibrinogen concentrate as versatile fibrin sealants amenable to a diversity of ambient thrombin, direct thermal, and spectrally induced thermal absorptive bonding in a broad range of fibrinogen/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 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. A still further objective is to determine and utilize the composition of the fibrinogen protein cryoprecipitated products obtained by a series of progressive recycling of the recovered supernatant plasma serum. Detailed Description of the Invention 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. 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 is also within the scope of the present invention. The fibrinogen concentrate of the present invention comprises a solids content made up of components in the molecular weight range of from about 18 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, plasminogen, prothro bin, thrombin, other proteins including and not limited to growth factors, and the like. The remaining 94% to 56% of the concentrate is water and other liquid components of plasma. The transition temperature varies for each of the component parts of the solids content of the fibrinogen concentrate. As the thawing time decreases, residual icing increases. The cryoprecipitate of the present invention may originate from mammalian plasma including and not limited 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, 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 remaining from plasma which has been passed through one cycle of cryofreezing, thawing, and centrifuging, may be reused or recycled to further prepare a cryoprecipitate 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, temperature, cryofreezing, thawing, and centrifuging. Mammalian plasma or cell plasma and modifications thereof with supplemental additions and inclusions of various kinds are within the scope of the present invention. Modifications and/or supplemental additions 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 for specific uses. These include such common modifications as (a) anticoagulants including ACD citrate or heparin, (b) anti-fibrinolytic agents such as aprotonin, and epsilon-aminocaproic acid, (c) coagulating agents such as calcium compounds, (d) viscosifying and thixotropic modifiers either naturally occurring or synthetic polysaccharides, mucopolysaccharides, and polygalacturonic acid, (e) bioadhesives in the form of mucoproteins or glycoproteins indigenous to plasma or serum or their synthetic analogs, (f) surfactants such as naturally occurring mucoproteins and mucopolysaccharides of the N- or O- substituted 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 microorganism activity. Supplementation of the fibrinogen 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 supplementing the fibrinogen concentrate after processing. In order to establish productivity, defined qualification tests, and standards lacking in the prior art, a novel and more efficient process engineering system was devised as herein described. A substantially higher level of solids content within the fibrinogen concentrate was achieved by applying a controlled thermal drift throughout the integrated cryoprecipitation, thawing, and centrifuging steps. In addition, the overall processing time is also shortened over that described in the prior art. The qualifications provided by the product of the present invention, serve as a basis for specifications for clinically safe and effective enhanced fibrinogen concentrate products for large scale production from pooled plasma and for limited small scale lots of enhanced autologous fibrinogen concentrates from patients in view of the prevalent risks of viral infections, notably numerous forms of hepatitis and human immunodeficiency virus (HIV) , from pooled or single-donor sources. The process for producing a fibrinogen concentrate from mammalian plasma comprising freezing said plasma to a temperature of at least about -20°C for less than four hours, thereby producing frozen plasma; thawing said frozen plasma at a temperature of at least about 4°C 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 concentrate having about 6% to about 44% solids content is also within the scope of the present invention. The freezing step of the present invention subjects the plasma to a temperature below the freezing point of the plasma. The temperature for freezing mammalian plasma in the process of the present invention is at least -20°C, more preferably about -20°C to about - 120°C, even more preferably -80°C. Freezing may be performed for less than about four hours, more preferably about 0.5 hours to about 4 hours, even more preferably about 1 hour. At these temperatures, the plasma is frozen to temperatures which compact the concentrate 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 temperature, more preferably from about 4°C to about 10°C, even more preferably about 4°C. The thawing step of the present invention is performed for a time range of from about one hour to about 30 minutes. The time and temperature of the thawing step of the process of the present invention are performed such that from about 5% 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 cryoprecipitates are thawed and then centrifuged to separate specific fractional products generally in a closed system to avoid environmental contamination using blood collection kits in conformity with standards prescribed by the American Association of Blood Banks. In bulk larger scale processing of pooled lots of blood, appropriate form and means of containment against environmental contamination are likewise provided for in similar processing stages or steps of cryoprecipitation, thawing, and centrifugation. Containers for use in the 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' 1 ). Cryoprecipitation is used as the initial, principal means of separating fibrinogen in preference to the previously mentioned precipitation alternates. It is minimally chemically reactive or disturbing to the intricate native configuration of the plasma proteins. Unlike lyophilization, cryoprecipitation does not remove water from the plasma. Accordingly, hydrogen bonds are not broken in the cryoprecipitate 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 temperature-time precipitation along with the thermal drift in relation to cooling rates and thawing rates. Considering the presence of hundreds of protein components in plasma of varying rates of insolubilization to the cryogenic temperature and the reverse of re- solubilization, the thermal drift in each direction provides the arena for limiting the nature and constitution of the fibrinogen concentrate. Cryoprecipitation is therefore a preferable means of producing the fibrinogen concentrate over the alternative of precipitation with adjunctive non- physiological chemical precipitants such as saturated salts, low molecular organic fluids or organic compounds that are suspect of imposing major physical, conformational changes in the molecular form and shape of the fibrinogen structures (Doolittle, 1975a) . Although cryoprecipitation is not in itself without some imposition of structural changes, it can be reasonably conjectured that the transition to and from cryogenic temperatures through the icing stage under restrictions of the temperature-time kinetics avoids the potentially drastic chemical environment on the extremely sensitive component chain linkages and their resistance to fibrinolysis (Doolittle, 1975b) . However, as is generally the inevitable phenomenon of continued chemical activity in the cryogenic state (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 concentrates. A critical feature of the invention is the discovery of cryoprecipitation and its hitherto nonobvious effects on yield and components of the cryoprecipitated fibrinogen concentrates. None of the currently available methods and preparations are acceptable for immediate clinical autologous usage within 2 to 4 hours. Following the cryoprecipitation process step, thawing is the next essential and critical component of the process of preparing the cryoprecipitate of the present invention. During thawing, the solid heterogeneous crystalline-like mush transforms into two phases of a viscous fluid with a glacialized homogeneous solid ice progressively melting with the thermal drift at thawing. The thermal drift is critical to the concentrate yield, the solids concentration, and the distribution of the numerous proteins through the solidus - liquidus equilibrium temperature. During thawing/de- icing, the frozen solid plasma releases the insoluble fibrinogen and innumerable associated proteins that are important for fibrinogen concentrates or more properly termed fibrinogen protein concentrates depending upon the attained purity of the fibrinogen concentrates. The latter is the ratio of the fibrinogen associated proteins which can be regulated by the thermal drift of the 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 mucoproteins, chemically known as glycoproteins which are indigenous to fibrinogen, and are also intended to be retained as much as possible within the purview of the present invention. The thawing is readily evident from the progress of de-icing and thereby regulated by selected time and temperature. Thus, thawing retains the useful and/or valuable plasma proteins natively associated with the complex structures of fibrinogen. By applying a specified critical control at the de-icing or thawing stage of the solid cryoprecipitate to the liquid watery state, throughout the time of continued thermal drift to and from cryofreezing, the new processing system results in considerably higher yields and solids content of the fibrinogen protein concentrate with a diversity of the associated useful protein contents. Thermal drift refers to the temperature differences between the external thermal exposure and internal thermal plasma states during the three processing stages of cryoprecipitation, thawing, and centrifugation. The overall process efficiency is thereby markedly and unexpectedly increased and processing time considerably shortened from the starting plasma to the separated fibrinogen concentrate. The retention of the associated proteins is highly dependant upon the thermal drift from the cryogenic state 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 continuation of the temperature time frame of thermal drift but with the minimal centrifugation time frame and with specified gravitation force at stated revolutions per minute (RPM) . As discovered for this application and indicated hereinafter, the need for minimizing the temperature time thermal drift presents a critical process intermediary 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 equilibrium transition temperature is a temperature at which, for each component of the solids content of the fibrinogen concentrate, the solid (i.e., ice or frozen) phase and liquid phase of a component are in equilibrium. For example, the solidus- liquidus equilibrium transition temperature for water is the temperature at which ice and liquid water exist in a percent ratio of about 50:50. Residual icing refers to the amount of components in the solid phase, i.e., ice, as compared to the components which have passed through the transition temperature into the liquid phase. Thawing permits each of the component parts of the plasma to reach a transition temperature such that the components pass from the solid phase to the liquid phase. By controlling the solidus - liquidus transition with time and temperature in the thawing step, the residual icing is thereby controlled. Control may similarly be established where thawing and centrifuging occur at the same time. Residual icing appears in the form of ice. In examples set forth herein, test tubes were used as a containment such that residual icing formed as ice plugs. Residual icing = weight or volume of ice plug x 100 weight or volume of initial plasma Following determination of the weight of residual 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% residual icing. Centrifugation is performed to produce a fibrinogen concentrate 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. EXAMPLE I This Example demonstrates the superior productivity, process efficiency, and product qualifications in the enhanced fibrinogen concentrates of the present invention. The new and enhanced fibrinogen concentrate products, referred to as Product B, are produced by controlled temperature-time thermal drift through the solidus - liquidus equilibrium transition from cryoprecipitation to centrifuged concentrate as described in Application Serial No. 07/562,839. The resulting products, summarized in Table 1, are provided with essential specifications and test methods hitherto not made known or available by the prior art, for assured safe and effective standards for clinical applications. A conventional fibrinogen product, as disclosed by Dresdale, A., Surgery, 1985, 67:751, is represented by Product A. Product A. Four aliquots, 40 ml each, of fresh frozen plasma were cryofrozen at -80°C for 12 hours followed by thawing at 4°C 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 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 cryofreezing was 1 hour and slow thawing was performed for 1 hour at 37°C, 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 the valuable associated plasma proteins into the liquidus phase. A comparison of the two respective fibrinogen concentrate products is summarized in Table 1 reflecting the numerous distinctive and surprising features of superiority of the concentrate Product B over that of prior art Product A with substantial advantages in productivity, process efficiency, and product qualifications for surgical tissue bonding are evident in Product A. Productivity and Efficiency Product yields - Solids Assay. Table 1 summarizes and compares the productivity in terms of dry solids of the controlled thermal drift Product B fibrinogen concentrate with that of the typical prior art Product A on a ratio (B/A) basis. The controlled thermal drift Product B provided a concentrate 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. Clottable Fibrinogen Assay. This assay of productivity is of prime importance as a qualification for effective and reliable surgical tissue bonding for several reasons. First, the assay is a definitive item of product specification related to controlling the thermal drift from cryoprecipitation to centrifugation. Second, the assay affects and relates to preemptive handling 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 protein components of the fibrinogen concentrate 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 fibrinogen of the concentrate products were used: 1. The clinical photometric measurement of turbidity of well-dispersed clotted fibrinogen induced by the conventional Ellis-Stransky thrombin-calcium chloride activation. This assay is useful for relatively low plasma levels of fibrinogen adaptable to high solids concentrates 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 fibrinogen 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- 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 concentrate, was used in this and other Examples, in at least two serial washes of the precipitated clotted fibrinogen concentrates. The washes were then vortexed to disperse the aggregate clots, centrifuged to firm sedimentation, decanted, and finally dried to constant weight at 80°C, usually in one hour, as described in Application Serial No. 07/562,839. The results shown in Table 1 demonstrate the enhanced clotted fibrinogen 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 embodiment of Product B, still higher yields of the clotted fibrinogen were attained and enhanced by higher ratios of clotted fibrinogen and residual plasma protein in the fibrinogen concentrate. Residual Proteins Assay. This assay is based on the difference between the dry solids yield, Table 1, and the clotted fibrinogen 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 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. Product efficiency. Taking into consideration the important processing engineering component of time. Product B was prepared in approximately one-seventh elapsed time (1/17.1) of Product A. Qualification Testing Of equal importance comparable to productivity and process efficiency is the qualification testing of essential product performance features such as easy handling, dispensing, and effective adhesive bonding of 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, several special preemptive qualification tests were devised as indicated in Table 1. Preemptive refers to testing for measured physical constants, notably viscosity and adhesive bonding using ex vivo collagen chamois substrate model in order to qualify for ensuing in vivo animal testing. Viscosity Test. The relatively low solids contents of fibrinogen concentrates 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- chilled, slightly thickened state. Despite published clinical reports referred to herein, the lack of viscous, adhesive tenacity accounts for the lack of clinical acceptance of fibrin sealants. A novel and practical means for measuring viscosity of the cryoprecipitates was devised relative to glycerol at 99% wt (1150 centipoises, at 21 ± 0.2°C, see Hodgman, CD., 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 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 enhanced viscosity. Bonding strength. A preemptive in vitro qualification test was devised for testing the native bonding quality of the fibrinogen concentrate emanating from the associated native plasma bioadhesives, notably the chemical variants 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 hydroxyproline binding sites available for either passive adhesion or thermally energized bonding. The distinction between passive adhesion and thermal adhesion or spectrally energized bonding, is not necessarily 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 mechanisms of measurable adhesive strength, in terms of force, and extension to failure or rupture. Passive adhesion is a particularly important quality in biomedical applications for initial tissue adherence of fibrinogen which in a pure state of itself is non-adherent on the scale of contact adhesion. Spectrally energized bonding is the attainment of measured bonding strength (force and extension) resulting from the application of absorbed energy, externally 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 thermally energized bonding is the result of specific or varied mechanisms 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 thereby elevating a proportionate share of the adhesive strength approaching the strengths of energized bonding. The preemptive dependence on the ex vivo chamois on tensile bonding testing, before undertaking in vivo animal testing, is fortuitous on several accounts for 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 provides practical evaluation in terms of tensile break force, a broad range of static and dynamic testing, and assessment of the nature of energetic bonding and direct adhesion. The chamois testing is considerably less expensive than animal testing, and capable of reasonable correlation to animal testing. Hence dependence 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- calcium activation of fibrinogen to fibrin. Three inch long by half-inch wide strips were cut laterally at the mid-point. These strips were rejoined at the cut with fibrinogen concentrates for the first stage of viscous adhesive bonding by the associated mucoproteins. The 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 1 to 2 drops (0.025 to 0.05 ml) of solution comprising 5 to 25 NIH units of thrombin in 40 mM calcium chloride per one ml of fibrinogen concentrate. The rejoined mid-cut butted edges of the test strips were placed in a 100°C oven for 30 minutes to activate maximal thrombin bonding, then cooled to room temperature for at least 30 minutes before testing for 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 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 minute. Similar specimens, having two pieces of chamois bonded by the cryoprecipitate of Product A and thrombin were prepared. The resulting tensile bonding strength was determined from the dimensions of cross sections of the bonded transverse area of the cut half-inch width multiplied by the measured thickness of each chamois test specimen 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, expressed as lb-f/in-w and second, as the corresponding 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 sensed qualitatively 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 systems in restoring the biomechanical integrity, the tensile break 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 vivo animal tests. The results summarized in Table 1 clearly demonstrate the exceptionally superior bonding strength with the enhanced solids content of the fibrinogen concentrate produced 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 superiority of elongation to break by a significant ratio (B/A) of 2.1/1 over that of Product A. Still further enhancements are made evident in succeeding Examples of the preferred embodiments. The chamois bonding test is a highly appropriate 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 found to be reactive to a variety of bonding systems described in this and ensuing Examples. Although varying considerably 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 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 efficiency and product 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 tissue bonding, referred to in surgery as tissue approximation or as anastomoses, and in turn for approved clinical trials and use. Table 1 Comparison of Productivity and Product Qualification Product A Product B B/A Prior Art Example 1 Initial Plasma 40 40 N/A Volume (ml) Concentrate 0.515 1.141 3.3/1 yield (grams) % solids content 6.24 12.4 2.0/1 dry solids 32 144 4.5/1 content yield (mgms) Clotted 28 121 4.3/1 fibrinogen yield (mgm) residual 4 23 5.8/1 proteins* (mgm) lapsed time A/B 16.3 2.3 7.1/1 ratio (hours) relative 26.7 83.3 3.1/1 viscosity** (centipoises) bonding strength-chamois, passive thrombin activated Tensile break 0.83 5.66 6.8/1 force (lb-f/in-w) % 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*** *Calculated, yield dry solids (c) minus clotted yield (d). **Relative to glycerol standard 1150 centipoises RT, based on force through clinical syringe 20 G 1 1/2 hypodermic needle. ***Regain of tensile break force and elongation to that of the control non-cut chamois stock material. EXAMPLE II Example II provides a preferred embodiment of fibrinogen concentrates made to at least 30% solids content by the controlled temperature-time thermal drift process. It also demonstrates increased productivity with several stages of repeated cryofreezing of recycled centrifuged plasma for recoverable fractions of fibrinogen concentrates comprising the essential, useful and valuable, symbiotic associated proteins. Also, it demonstrates that with the controlled temperature-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 demonstrated with substantial increases in the productivity of the fibrinogen and associated plasma glycoproteins as summarized in Table 2, on productivity and qualification tests far exceeding that shown in Table 1. Product - Controlled Thermal Drift. Four aliquots, 38 ml each, of fresh frozen plasma having initial plasma solids of 9.74%, from extended storage at -20°C (not over three months) containing 0.025 ml Kefzol (Lilly) antibiotic solution (1 gm in 2.5 ml sterile water) and 20 mgm of epsilon-aminocaproic acid (EACA) were placed in sterile 41 ml polypropylene centrifuge tubes and cryofrozen at -80°C for 1 hour. Without a separate step of thawing, the cryofrozen aliquots were placed directly in the 4-place Du Pont HB-4 swinging ^ ucket of a Du Pont RC-5C Sorvall Superspeed Centrifuge. The centrifuge was pre-set to 14°C for the centrifuging time of 32 minutes at 8000 xg. The selected temperature-time combination with centrifugal force - 28 - thereby controlled the concurrent thawing through the solidus - liquidus transition to a residual solid plug of ice of 15 gm, corresponding to approximately 40% residual icing transformed from the crystalline cryofrozen mush during' the centrifuged thawing. The sedimented cryoprecipitates of the four 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 the decanted supernatant 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 each of the three intended demonstrations of substantial increases in productivity of fibrinogen and its associated plasma proteins, process efficiency with elimination of the separate thawing step, and recycling of supernatants for enhanced productivity. It is particularly noteworthy that the three Fractions I, II, III, the last two of which were produced by recycling the successive supernatants starting from Fraction I, resulted in substantial increments of additional concentrate yields. Starting with Fraction I with an initial concentrate 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 concentrate with the associated plasma proteins. Because of the inordinate variability of human plasma with innumerable components, over some 1000 protein configurations, with a wide range of dry solids contents, the foregoing productivity and ensuing quality test specifications can be expected to reflect corresponding variability in yields and quality test results. The same can be expected for the inordinate combinations of the applied variations in temperature, time, and centrifugal in the course of the cryofreezing, thawing, and centrifugation. Clotted Fibrinogen Assay and Yield. The productivity with the shortened controlled thermal drift time by the elimination of the thawing step is also evident in the yields of the clotted fibrinogen 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 decreases with each ensuing Fraction, the corresponding 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 the controlled thermal drift of this Example indicates that all of the fibrinogen and associated plasma protein would be exhausted in the next Fraction IV and Fraction V. The assay of the clotted fibrinogen concentrate content is determined by the ethanol precipitation method 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 concentrate solids yield expressed in milligrams. The corresponding remainder of non-clotted product yield constitutes the cryoprecipitated 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 with the initial 470 mgm yield in Fraction I, representing 28.8% of the solids yield, the yields increase progressively with the successive recycled Fraction II, 1015 mgm, and Fraction III, 762 mgm, to the corresponding 46.0% and 64.0% composition in the cryoprecipitated concentrate. Qualification Tests Qualification testing is an important feature of providing specific tests related to the intended 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 adequate initial adhesive strength that may be passive or 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. Viscosity. 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 significantly increases the relative viscosity from 83.3 to 154 centipoises. An increase in the viscous adhesive quality, particularly important for initial sticking to tissue surfaces, is thereby provided. Substantial increases in viscosity are also attained in the succeeding Fractions II and III. The viscosity qualification test devised in this Example is a highly important test used to ascertain the shelf life of stored concentrates in relation to either increase or decrease of viscosity due to potential molecular changes involving clotting or fibrinolysis controlled by inclusion of appropriate preservatives and antibiotics. Bonding Strength. Chamois - passive thrombin-calcium system. 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 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 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 with 5.11 and 5.65 lb-f/in-w bond strength and 20.2% and 20.5% regain to control tensile strength regain are considered acceptable for in vivo animal testing. For more rapid tissue bonding, particularly in a matter of seconds, the passive adhesive bonding can be augmented with thermally activated intermolecular mechanisms of cross-linking, disassociation, polymerization, etc. using spectral penetration or absorption with endothermic heating as demonstrated in the ensuing examples. Bonding strength. Microwave bonding. Microwave penetration of the interface between the fibrinogen concentrate and the tissue substrate provided a convenient means for ascertaining the supplemental contribution of energized molecular motion for supplementing the passive adhesive bonding. The preparation of the test specimens using the three Fraction concentrates applied directly to the joining edges of the chamois specimen is described in Example I. The specimens were then placed in a microwave field of a household unit at a microwave frequency of 2420 Mz, powered by single phase 120 VAC 60 Hz of 900 Watts, at exposure 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 demonstrating 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 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 succeeding example of laser activated bonding of the three Fractions is provided. The chamois bonding also serves as a useful means for evaluating and comparing the efficacy of fibrinogen prepared by precipitation with ethanol directly from plasma at room temperature and reconstituted to 36% 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 force or approximately 1/24 of that attained with the 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, of the essential residual native adhesive plasma proteins. Bonding Strength. Chamois - laser activated bonding. This laser activated bonding uses indocyanine green dye (Cardio-Green, Becton, Dickinson and Co., Cockeysville, MD) having a maximum absorption at 805 nm with an extinction coefficient of 2 x 10 5 nr'cm '1 . 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 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 concentrated 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) coupled to a hand held focusing optic with a beam diameter of 2 mm and directed at distance of 4 cm from the concentrate 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, 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 dye absorption laser bonding for the three Fraction concentrates revealing higher bonds strength with Fraction II compared to Fraction I and a lower bonding strength with Fraction III. There appears to be a dependency of the bond strength upon some optimum ratio of clotted fibrinogen to residual proteins, such as listed in Table 2. However, the range of the ratios with the three Fractions qualifies all three ratios for effective in vivo animal tissue. In vivo Animal Tissue Testing. Enhanced fibrinogen concentrates, 12% to 40% dry solids content, prepared according to the controlled thermal drift process. Minimal chamois thrombin activated bonding strength of 1.2 force-pounds per inch width, was tested 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 healing to the complete restoration of the biomechanical integrity. The incisions were made under anesthesia in duplicate on either side of the spine, usually with a random pairing of different preparations of concentrates, pairing concentrates with sutures, and pairing the three bonding methods of opposite sides of the spine. The retrieval specimens 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 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 midincision restraint was sutured as a safeguard against early rampant rupture due to chance 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 nonincised specimens for assessing each of the three means of tissue restoration. Table 2 summarizes the results of the extended healing of the fibrinogen tissue bonding comparing the passive thrombin activation bonding with that of the laser spectral absorption and in turn with that of 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 28 to 90 days the thrombin and laser bonding modality are statistically equivalent, but attain higher rupture strength than the sutured modality at 90 days attain the fully restored, healed biomechanical integrity. Table 2 Enhanced Productivity and Qualification Tests Processed I II* III* Aggregate Fractions Initial 38 X 4 36 X 4 34 X 4 152 Plasma Volume (ml) concentrate 4.67 5.98 3.79 14.48 yield (grams) % solids 34.9 36.9 31.4 N/A content Solids 1630 2207 1190 5027 yield, dry (mgm) clotted 1160 1192 428 2780 fibrinogen 71.2 54.0 36.0 N/A (mgm) % residual 470 1015 762 2247 protein 28.8 46.0 64.0 N/A (mgm) % fibrinogen/ 2.47/1 1.17/1 0.56/1 N/A protein ratio lapsed time 1.5 3.1 4.6 N/A (hours) Relative 154 131 125 N/A viscosity ** (centi¬ poises) Bonding Strength - Chamois, Passive Thrombin Activated RT, 24 Hrs. tensile 7.45 5.11 5.65 N/A break force lb-f/in-w % regain to 32.4 22.2 20.5 N/A control*** % Tensile 27 21 20 N/A elongation % regain to 41 29 31 N/A control*** Processed I II* III* Aggregate Fractions Bonding strength - chamois, microwave 2 seconds Tensile 8.5 9.54 8.18 N/A break " force lb-f/in-w % 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, laser dye absorption Tensile 2.62 1.73 1.14 N/A break force 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-f/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 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 regain * Recycled supernatant from preceding processing stage. ** Relative to glycerol standard, 1150 centipoises, forced through clinical syringe, 20 G 1 1/2 hypodermic needle. *** Regain of tensile break force and elongation compared to control, non-cut chamois stock material. **** Regain of tensile break force compared to that of control, non-cut adjacent dorsal skin. ret'l = retrieval ref. = reference EXAMPLE III The purpose of this example is to prepare cryoprecipitated fibrinogen concentrates from a series of mixtures of pooled plasma with added albumin. Albumin was included to supplement fibrinogen concentrates with the adhesive glycoproteins and the low molecular weight pre-albumins that contain valuable factors for cell growth for healing of incised tissues bonded with the fibrinogen concentrates. It is evident from the 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 and effective levels of adhesive tissue bonding as well as substantial productivity of a clinical product. Preparation of Concentrates The controlled thermal drift process described in Example II was applied to a progressive series of human plasma - human albumin compositions using unused portions of collected pooled plasma of varying refrigerated storage times up to 11 months at -20°C. The pooled plasma was supplemented with clinical, U.S.P. grade albumin (human 25 solution) in a progressive series of proportionate levels of 0%, 10%, 20%, and 40% admixture. Table 3 summarizes the principal specification items wherein the albumin supplemented not only the productivity of the process but also replaced a significant portion of the plasma for the qualification tests for adhesive bonding. The plasma was cryofrozen at -80°C for about one hour. Thawing and centrifugation were performed simultaneously at 14°C for 32 minutes at 8000 xg. 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 marked increase in the solids yield, also see Table 3, over that of the control. This indicated a significant and surprising yield of clotted fibrinogen along with associated native proteins for their adhesive value. Thus, in a single production stage, without recycling the supernatants, the one single cryoprecipitated Fraction I of the admixtures with albumin is a novel and highly useful product for adhesive bonding. It is noteworthy, unexpected and surprising, that the supplementation with albumin induces marked molecularly associated cryofreezing with substantial solids concentration and yields of clotted fibrinogen 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 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 fibrinogen. Qualification Tests The admixtures of the human plasma with human albumin resulted in significant and consistent increases in the relative viscosity, shown in Table 3, thereby providing enhanced viscous contact, a quality of stickiness, to tissue substrates. In the chamois passive adhesion bonding strength, Table 1, using thrombin- calcium chloride activation, the albumin supplementation provided significant enhancement in tensile break force and elongation commencing at 20% to 40% level. Similar enhancement 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 Example II, considerable variations in the extent of the attained tensile break strength was made evident with the three different bonding systems each having optimal maximal enhancements of tensile break strength and elongation depending upon applied thermal or spectral 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. Table 3 Productivity and Qualification Tests Supplementation of Plasma with Albumin Plasma Albumin 100/0 90/10 80/20 60/40 (vol/vol) plasma (ml) 38 36.2 30.4 22.8 albumin (ml) 0 3.8 7.6 15.2 Productivity recycled fraction series Concentrate 1.333 1.614 1.845 1.762 yield (grams) % solids 31.5 34.8 34.0 36.3 content dry solids 420 562 627 640 yield (mgm) Clotted fibrinogen mgm 354 411 407 280 % 84.3 73.1 64.9 43.8 residual proteins* mgm 66 151 220 360 % 15.7 26.9 35.1 56.2 fibrinogen 5.36/1 2.72/1 1.85/1 1/0178 protein ratio Viscosity** 157 170 173 168 centipoises Bonding Strength - Passive Thrombin Activated RT 24 hrs tensile break 1.20 1.20 1.80 3.20 lb-f/in-w % 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 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 control*** ♦Calculated, dry solids yield (c) minus clotted 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 of the control non-cut chamois stock material. EXAMPLE IV The purpose of this example is to extend the albumin plasma supplementation with recycled supernatants throughout the repeat stage of controlled thermal drift from cryofreezing to centrifugation. This example also demonstrates increased productivity of the recycled Fraction concentrates for a wide range of useful fibrinogen/protein ratios. Preparation of Recycled 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 consecutive repeat stages. Table 4 summarizes the resulting principal specifications 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 pediatric and elderly cases. The plasma was cryofrozen at -80°C for about one hour. Thawing and centrifugation were performed simultaneously at 14°C for 32 minutes at 8000 xg. Productivity The admixture of 40 parts albumin to 60 parts of single donor plasma lot provided about 30% 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 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 vivo storage, notably with fibrinolysis and intermolecular associations. In this example, listing only the Fraction I productivity, the fibrinogen/protein ratio, showed approximately 75% higher proportion of clotted fibrinogen. These specifications of the productivity invoked substantial differences in useful quality as detailed in the ensuing section and further emphasized the inadequacy of the prior art in anticipating or predicting useful qualities. Qualification Tests The admixture with 40 parts of albumin provided a modest level of viscosity, substantially enhanced with successive recycling throughout all four Fractions attaining a maximum at Fraction III. This provided an 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 deceases in clotted fibrinogen to as low as 3.4%, provide substantial bonding strengths throughout the entire recycled Fraction series, see Table 4. It may be expected 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 ratio not only in instant or immediate but also on prolonged healing to complete biomechanical restoration in specific terms of regained tensile break strength and elongation. *Calculated, dry solids yield (c) minus clotted fibrinogen (d) . **Relative to glycerol standard, 1150 centipoises RT, 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. EXAMPLE V This Example demonstrates the productivity and qualifications of bovine fibrinogen concentrates product as the extension of the controlled thermal drift process to other mammalian plasma. Preparation of Concentrates The same procedures from cryofreezing to centrifugation as described in Example II were applied to bovine plasma. The leading Fraction I serves to 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 specifications of productivity and qualification tests using a commercial source of bovine plasma with an initial plasma solids assay of 14.3%. This example illustrated the general applicability of the temperature- time thermal drift process and the resulting enhanced fibrinogen concentrate products from different variants of cryoprecipitated types of viable plasma. The plasma was cryofrozen at -80°C for about one hour. Thawing and centrifugation were performed simultaneously at 14°C for 32 minutes at 8000 xg. Productivity 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 human plasma, and even higher with the successive recycled Fractions II and III. The successive series of Fractions demonstrate the consistent general trend of increasing clotted fibrinogen yields along with that of 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. Qualification Tests The leading qualification of viscosity increased substantially with the successive recycled Fraction series imposing a pronounced effect of the bonding quality. In the case of passive thrombin activated bonding the increase continued consistently from Fraction I to Fraction III. In the case of the microwave thermally 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 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 exposure time or a depletion of a bioadhesive protein component but with a useable and effective level of bonding equivalent to that of Fraction I. In this chamois bonding series, a reconstituted 36% concentrate of bovine fibrinogen, Type I-S of a commercial source, in Ringers lactate was bonded by microwave exposure for 2 seconds, see Table 5. The resulting tensile break force of 0.92 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 bovine was lacking in both the intrinsic adhesive quality and the response to thermal bonding incurred during the processing, presumably denaturation, from the native aqueous concentrate state to the dried dehydrated powdery form. In Vivo Animal Tissue Testing. The same dorsal incision bonding on Wistar rats as described in Example II was applied to the time-extended series of retrievals in the qualification tests for 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 fibrinogen concentrate on opposite dorsal sides of incisions for the initial critical period of 4 to 28 days of healing. The results indicate that the bovine and human fibrinogen 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 that of the control non-incised tissue. Table 5 Productivity and Qualification Tests of Bovine Fibrinogen Concentrates Bonding Strength microwave 2 sec tensile 7.63 6.48 6.68 N/A break force lb-f/in-w % 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 control*** Bonding Strength - chamois laser dye absorption 10 sec tensile 3.55 6.15 3.80 N/A break force lb-f/in-w % regain to 14.4 24.9 15.4 N/A control*** % 45.2 47.3 47.2 N/A elongation % 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, based on force through clinical 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. Modifications and Equivalents The herein described Examples of preferred embodiment, cryofreezing, thawing, and centrifuging, to produce enhanced viscoadhesive fibrinogen concentrates 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 embodiment Example II. For example, thawing and centrifuging may take place simultaneously. Such process modifications for adjusting the productivity, process efficiency, and qualification test specifications are described in the Application Serial No. 07/562,839. Modifications produce enhanced cryoprecipitated concentrates from about 12% to as high as 40% solids of useful and effective viscoadhesive 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- dissolving of cryoprecipitated plasma proteins into the liquidus 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 for modifying the native 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 the Examples is the progressive recycling of the spent supernatants to yield Fraction series of concentrates with an assay of progressive lowering of the fibrinogen/residual protein ratios but effective in ex vivo tissue adhesion. The Fraction series can be used to make composite admixtures to stated product specifications adjusted for solids content and/or the fibrinogen/native protein ratios where appropriate in specific types of tissue bonding or restructuring. The foregoing disclosures and descriptions of the qualification tests for, and accomplishing viscous adhesion and passive and/or spectral absorptive bonding may be appropriately modified to the degree of desired bonding strength. The latter would apply to some preferred minimal solids concentration standard between 12% and 40% or more handling in surgical application dispensed from syringe at a preferred range of viscosity. It may, by personal choice be other than the mid range nominal 36% solids used in Example II, either higher or lower. This also applies to the varying choice of the optimal fibrinogen/residual protein ratio depending upon the type of the anatomical tissue, for instance, from exterior skin structure to fine internal vascular or gastrointestinal to relatively thin, often of microscopic dimensions and delicate ophthalmic and neural sheath tissues. In this wide range of tissue structures, 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 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 to replace or augment suturing as a means of reducing 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 involving fibrinogen polymerization and spectral absorption with directed laser. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims: What is claimed is: 1. 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. 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. 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. 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. 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. 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. 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. The concentrate of claim 1 wherein said mammalian plasma is selected from the group consisting of human, bovine, porcine, rabbit, and equine plasma. 9. The concentrate of claim 1 comprising about 12% solids content. 10. The concentrate of claim 1 comprising about 24% solids content. 11. The concentrate of claim 1 further comprising albumin. 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. The concentrate of claim 1 f rther comprising components selected from the group consisting of anticoagulants, antifibrinolytics, coagulating agents, viscosity modifiers, bioadhesives, surfactants, antibiotics, and preservatives. 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. 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. The tissue adhesive of claim 8 comprising about 12% of solids content. 17. The tissue adhesive of claim 8 comprising about 24% solids content. 18. The tissue adhesive of claim 8 further comprising albumin. 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.

Inventors: LONTZ JOHN F, LONTZ, JOHN, F.
Applicants: FIBRIN CORP, FIBRIN CORPORATION
Application year: 1995
Main classification: A61K38
IPC codes: A61K38, A61L24, C07K14