CA1306583C - Pasteurizable, freeze-driable hemoglobin-based blood substitute - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present specification describes a process by which a blood substitute (hereinafter referred to as "HemoSafe") is derived from uniformly stabilized monomers and polymers of deoxyhemoglobin in its tight (T) conformation, with oxygen affinity similar to that of human blood. Two classes of HemoSafe are derived respectively from animal-hemoglobin and human-hemoglobin. HemoSafe (animal) differs from HemoSafe (human) in that it is free of polymers in order to reduce potential immunogenicity if used in man. Both types of HemoSafe may be derived in the following manner. The stabilized deoxyhemoglobins are converted to their carbonmonoxy derivatives (CO-HemoSafe) which are then stable under pasteurization conditions to render them viral disease transmission-free. CO-HemoSafes are stable for 2 months at 56°C in either the solution or the freeze-dried state. For transfusion CO-HemoSafes are easily oxygenated under sterile conditions by photoconversion yielding oxy-HemoSafe. In addition a transfusable met-hemoglobin derivative for treatment of cyanide poisoning, is derived by converting oxy-HemoSafe to met-HemoSafe.

Description

~3~)~S~33 This invention relates generally to the stabilization of biomacromolecules in unique conformational states, and utilization o~ their associated activities and functions for biomedical and biotechnological applications. More specifically, hemoglobin (~b), in aither of its natural conformations ttight Hb(T) or relaxed Hb(R)~, is stabilized and/or cross-linked to prevent dissociation and to achieve desired ligand-binding affinity. Processes and reagents are developed ~or the preparation of a family of new animal-hemoglobin, human-hemoglobin and genetically engineered hemoglobin-based substitutes for the oxygen delivery function of red blood cells, or "blood substitutes", and conversion of the oxyhemoglobin derivative to the met-hemoglobin derivatives for use in treating cyanide poisoning.

There are severe limitations on the use of blood as a transfusion fluid. These stem largely from the natural characteristics of red blood cells (R~C) and the danger of disease transmission.
Viral disease transmission by blood and blood products is a major problem in transfusion medicine. Screening to detect virally lnfected blood is cos~ly and not completely effective.
Of the available methods for inactivating viral infection in ~L3~ 51~

blood-derived pharmaceuticals, an industry standard is wet-heat pasteurization. RBC are not, however, pasteurizable.
Other limitations on ~he use of whole blood in transfusion include the rigorous storage requirements, short shelf-llfe, and complex immunologic characteristics of RBC. Hence, extensive research has been done to develop a cell-free, hemoglobin-based blood substitute through chemical modification of stroma-free hemoglobin.
As is well known, hemoglobin naturally comprises a tetramer of four subunits, made up of two alpha (~) and two beta (B) globin chains. The molecular weight of the tetramer is about 64 kilo-daltons, and each of the sub-units has approximately the same molecular weight. In dilute solution, tetrameric hemoglobin readily dissociates into half molecules of~ ~ dimers. The relatively low molecular weight of these dlmers is such that they are rapidly flltered from the clrculatlon by the kidney and lost in the urine. This results in an unacceptably short half-llfe (T 1/2) of approxlmately 2-3 hours.
Furthermore, hemoglobln without the protection of its red cell membrane loses its natural ligand diphosphoglycerate (DPG).
DPG normally lowers the oxygen affinlty of hemoglobln, so that, after lt ls lost, soluble hemoglobin binds oxygen more tightly and does not dellver oxygen to the tlssue as efficlently as do red blood cells. For these reasons, stroma-free hemoglobln is not suitable for use as a blood substitute.

~306~3 These defects have been addressed in the past by covalently attachlng DPG analogues such as pyricloxal-5'-phosphate (PLP), to hemoglobin in its deoxy-state Hb( T ), yielding PLP~Hb which has a lower oxygen affinity approaching that of RBC. Accordingly, Greenburg (Greenburg et al., Surgery, vol. 86 (1979), pp. 13-16) explored the use of PLP-Hb with physiological oxygen affinity as a blood substitute. ~owever, without intramolecular cross-linking PLP-Hb, like SFH, dissociates into half-molecules and is rapidly excreted by the kidney. Furthsrmore, Hsia and co-workers (McGarrity et al., Journal of Chromatography, vol. 419 (1987)~ pp. 37-50) have shown that PLP-Hb is a complex mixture of modified hemoglobins containing between O and 6 moles of PLP per mole of hemoglobln, each of these subspecies having a different oxygen affinity.
DPG analogues have been shown to cross-link Hb intramolecularly. These cross-linkers are designed to bind specifically to the DPG-binding site of human hemoglobin. They include reagents specific for B subunits (Benesch et al., Biochem. Biophys. Res. Commun., Vol. 63, No. 4 (1975), pp. 1123-1129, Benesch et al. in Methods in Enzymology, Vol. 76.
Hemoglobins (1981) pp. 147-159 Academic Press) orC subunits (Chatter~ee et al., Journal of Biol. Chem., Vol. 261, July 25, 1986, pp. 9929-9937).
Cross-linking by these specific ligands generally gives low yield (<70%) and requires further purification of products.
Activated triphosphate nucleotides have been used simultaneously 13065~3 to cross-link hemoglobin and to occupy the DPG binding site, yielding ATP-Hb which was found to have lower oxygen affinity than PLP-Hb, and longer plasma retention time. (Greenburg et al., Pr~gress in Clinical and Biological Research, Vol. 122, Advances in Blood Substitute Research (1983), pp. 9-17, Alan R. Liss, New York; McGarrity et al., Journal o~ Chromatography, Vol 415, (1387), pp. 136-142. At 80-90~ blood rep~acement, ATP-Hb is extravasated into pleural and peritoneal spaces, so that it may not be safe to use at high levels of blood replacement.
Intermolecular cross-linking (polymerization) of PLP-Hb by a nonspecific cross-linker [e.g. glutaraldehyde (GA)] was introduced to prolong plasma retention time (Bonhard et al., United States Patent 4,136,093, Jan. 23, 1979). The resulting polymerlc PLP-Hb (14 g Hb/dl) had physiolog~cal oxygen-carrying capacity (up to 20 cc 2 per dl) and iso-oncotlc pressure.
However, the polymerization is incomplete and its components have heterogeneous oxygen affinity.
Yet other groups have attempted to prolong plasma half-life by conjugating PLP-Hb with polyethylene glycol (Iwasaki et al., Artificial Organs, Vol. 10, No. 5, (1986), pp. 411-416), inulin (Iwasaki et al., Biochem. Biophys. Res. Commun., Vol 113, No. 2 (1983), pp. 513-518), and Hb with dextran (Tam et al., Proc. Nat'l. Acad. Sci., USA, Vol. 73, (1976), pp. 2128-2131).
The heterogeneity of the products and their h~gh oxygen affinity were again drawbacks.

~ 3~t~5~33 Polymerization of Hb using a variety of divalent cross-linkers yielding poly Hb as a blood substitute has been d~scribed in the prior art. The drawbacks of this approach are excess polymerization (90-~ of the product is greater than 150 kilo-daltons in size), variable oxygen affinity, complex reaction schemes, and biological incompatibility of the cross-linkers used (e.g. divinyl sulfone, a potential carcinogen - US Patents ',7~ GC~ OO
~- -4,001,~00; 4,001,401; 4,053,590). In a follow-up to this approach, the same family of divalent reagen~s has been used to produce intramolecularly cross-linked Hb. Again, divinyl sulfone has been shown to give a product with variable oxygen affinity and unspecified composition (US Patent No. 4,061,736).
It is an ob;ect of the invention to provide novel blood substitutes and processes for their preparation.
It is a further obJect of this invention to provide stabilized and pasteurized hemoglobin useful as a therapeutic agent in transfusion medicine, said pasteurizatlon rendering the hemoglobin substantially free of transmissible infection agents.
A further object is to make available cross-linking agents and processes which cause hemoglobin to become intramolecularly cross-linked in either of its natural molecular conformations, (tight (T) or relaxed (R)), such that conformation is retained in the cross-linked product, which is then said to be conformationally stabilized. Such conformational stabilization is applicable to biomacromolecules in general.

13065~3 The present invention is based upon a novel manner of stabilizing hemoglobin in its tetrameric form, which yields a hemoglobin tetramer not only stabilized against dissociation into dimers, or monomers, but is also stabilized in either of the natural conformations of Hb, namely tight (T) as normally assumed by deoxyhemoglobin, or relaxed (R) as normally assumed by oxyhemoglobin. Instead of using DPG analogues to stabilize and cross-link Hb(T), the products of the present invention are stabilized against both dissociation and conformational change by covalent chemical linkages between globin chains of the respec ive sub-units. Whatever the conformation of the starting tetrameric hemoglobin, T or R, ~hat conformation is retained in the stabillzed product according to the invention.
The ability of Hb to shift between conformations i5 reflected by the ~ill coefficient (n). A coefficient of 2 or more indicates cooperative oxygen binding between subunits through conformational change, while a coefficient approximating to 1 indicates that Hb is locked in the T or R conformation. A
Hill coefficient approximating t~ 1-1.5, indicating complete stabilization and the absence of co-operativity in oxygen binding, is a distinctive feature of the present invention.
A further distinction of the present invention over the prior art is that in the prior art, stabilization of hemoglobin is accomplished by linking globin chains of the sub-units at specific sites to make up the hemoglobin tetramer. These _ 7 _ ~3~5~3 products are stabilized only against dissociation of the tetrameric units into dimeric sub-units, i.e. cooperative binding of oxygen is retained (Hill coefficient n~). Th~ present invention, in contrast, stabilizes tetrameric hemoglobin not only against dissoci~tion but also agalnst conformational change, i.e.
no cooperative oxygen binding (~ill coefficient n~1-1.5). This feature of conformational stability is unique to the products of this invention.
The conformationally stabilized tetrameric hemoglobin of the present invention shows a number of significant advantages. For example, the very fact that it is stabilized into the T-conformation, and remains in that conformation in solution, confers on the product an oxygen affinity approximating that of natural red blood cells. The products of the invention are therefore substantially equivalent with red blood cells in respect of thelr oxygen uptake at the lungs. Another important feature is their ability to deliver oxygen to the body tissues.
Under the oxygen partial pressure experienced in the body tissues, the T-conformational stabilized products of the invention will release a substantially greater quantity of oxygen to the tissues than allosterically cooperative, non-conformationally stabilized hemoglobin products. This high oxygen release to tissue becomes very important for example, when transfusing to the tissue-injured trauma patient, as commonly encountered in emergency medicine.

~306S83 A very signiflcant contrast of the present invention with the prior a~t is that the stabilized hemoglobin (HemoSafe I), according to the invention is readily produced in high yield, (at least 95%) in a single reaction step, substantially free from residual unstabilized hemoglobin. This does not require further purification, so that there is very little per cent formation of low molecular weight dimeric product in solution, even without subsequent purification to remove residual unreacted material.
Accordingly, the conformationally stabilized Hb in the form of tetrameric units (hereinafter sometimes referred to as HemoSafe I) has a plasma half-life of about three times that of stroma-free hemoglobin and so may be well suited for use as a blood substitute in emergency situations. In addition, this stabilized tetrameric Hb(T) is eminently suited to the preparation of other novel hemoglobin-based products. Tetrameric Hb(T) can be linked together to form a polymerized hemoglobin, (poly Hb(T) or HemoSafe II) useful as a blood substitute and in most circum-stances preferred as such to the stabilized tetrameric product on account of its increased molecular weight, conferring longer plasma half-life. The stabilized tetrameric Hb can be covalently linked to a second bio-polymer to form a conjugate (HemoSafe I-conjugate), useful as blood substitutes. In all of these conversions, the hemoglobin tetramers retain their specific conformation (e.g. Hb(T)). The product can be lyophilized for extended storage.
Moreover, and of great practical significance, it has been found that the conformationally stabilized heme-containing 9 ~ 3q:~S~33 tetramers of the present invention can be treated to avoid oxidatlon of the iron from the ferrous to the ferric state, e.g.
by presence of o~yg~n displacers such as carbon monoxide or nitric oxide, or oxyyen scavengers such as sodium dithionite, and then pasteurized to destroy any contaminating viruses. The produ~ts of the invention are sufficiently ~table to withstand the heating required, to 60C for ten hours at least, for pasteurization without denaturing of the protein or decomposition of the product. This process can be applied to pasteurize the tetrameric, e.g. C0-HemoSafe I, the polymerized, e.g. C0-sl ~
HemoSafe II and the ~ugntcd-C0-HemoSafe I-con~ugate. Then these C0 HemoSafes can be readily reconverted to oxy-HemoSafes whiah are suitable for transfusion as blood substitutes, all without significant loss of their conformational functional and structural integrity.
Further, the products can be oxldized, wlth or without polymerization or conjugation, to met-hemoglobin products, (i.e.
met-HemoSafes) effective as cyanide scavengers and usable for such purposes prophylactically.
Thus, according to the present invention, in a broad aspect, there is provided a hemoglobin product consistlng essentially of tetrameric hemoglobin units stabilized against dissociation into dimers and stabilized against conformational change between the T-conformation and the R-conformation upon formation of aqueous preparations thereof, said tetrameric units having covalent ~306583 chemical linkages between globin chains of the sub-units to effect the stabilization.

In the preparation of products of the invention, novel reagents, reaction conditions and processes for stabilization of a uniform composition of deoxy-Hb (Hb(T)), and for protecting it from instability under freezing or up to a temperature of 60C, have been developed to achieve pasteurization, lyophilization and uniform physiological oxygen affinity of the finished products.
Conformation S~ecific Stabilizers and Cross-linkers (CSSC):

. . _ . _ . _ _ _ The preferred method of achieving the desired conformational and intramolecular stabilization of the tetrameric hemoslobin units according to the invention is by reaction with one or more of a class of reagents called conformation-specific stabilizers and cross-linkers (CSSC). Most commonly, these are dialdehydes or polyaldehydes. They react with primary amino groups on the globin chains, to form Schiff base linkages, which can subseguently be reduced to secondary amine linkages. In tetrameric hemoglobin, there are appropriately located primary amine groups on the~ -chains and the ~-chains which will react preferentially and to all intents and purposes selectively with dialdehydes and polyaldehydes to form the desired covalent linkages for stabilization.
One example of a dialdehyde for use in the present invention as a CSSC is glutaraldehyde, (GA), when used under proper conditions. Reaction of hemoglobins with GA has been reported previously. These previous processes, however, have 1306~3 been conducted under conditions which did not lead to essentially complete stabilization of conformation of the tetrameric units, and did not lead to a product consisting essentially of stabilized tetramers. As previously used, glutaraldehyde is a non-specific reagent, and causes covalent intermolecular linking between tetramers to form polymerized hemoglobin faster than it forms intramolec~lar links between sub-units of a tetramer to stabilize it. The result is a mixture of stabilized tetramer, unstabilized tetramer, and stabillzed polymer of wide molecular weight distribution, each component having different oxygen affinity.
In the present invention, hemoglobin is reacted with GA
using a low concentration of hemoglobin (i.e., 1-4 g/dl) and a high molar ratio of GA to hemoglobin (i.e., from about 6:1 to about 60:1). Under these conditions, at least 95~ and commonly at least 98% stabilization of the Hb monomers in tetrameric form is achieved in the polymerized hemoglobin. An essentially completely conformationally stabilized and polymerized product is thereby achieved. The stabilized polymeric form can then be carbon monoxylated for pasteurization.
If animal hemoglobin-based blood substitutes are to be used in man, formation of oligomers and polymers should be prevented because of their antigenicity.
During the process of fixing the Hb in the monomeric form, Hb(T), with GA, there is strong tendency towards polymerization.
Therefore, a preferred embodiment of the invention employs 130~5~33 different CSSC reagents and reaction conditions in the stabilization of hemoglobin (Hb). Use of these CSSC compounds can achieve stabilization of the Hb monomers, minimum dimer formation (<5~) wi-th no detectable polymers, and, as well, the different CSSC compounds can generally be ~sed to stabilize other macromolecules in the conformations chosen, with minimum polymerization, if desired. Such CSSC compounds, by stabilizing macromolecules in unique conforma~ional states, permit the preservation of the activity and function of the bio macromolecule. However, use of CSSC compounds can additionally permit manipulation of the reaction conditions deliberately to link macromolecules to each other (polymerization), and alternatively to conJugate them to a dlfferent macromolecule, e.g. Inulln. Specifioally, using deoxy hemoglobin in its tight (T) conformation (Hb(T)), with such a CSSC compound as the linking reagent, the reaction conditions can be manipulated to allow polymerization of the stabilized Hb(T) monomers, if desired, as well as linking of stabilized Hb(T) with different macromolecules. Such CSSC compounds can also be used to stabilize and to polymerize hemoglobin derivatives, e.g. PLP-Hb, depending on reaction conditions, and additionally, to link such derivatives to different macromolecules.
The CSSC compounds used in the present invention are generally dialdehydes or polyaldehydes. One specific example thereof is glyoxal, of the formula HOC-COH. Other specific examples are benzene dialdehydes (ortho, meta and para), although - 13 - 13~583 aromatic cross-linkers are less preEerred because of the risk of immunogenic reaction from the residual aromatic groups. Most preferred as CSSC compounds are the aldehydic products produced by oxidative ring opening reactions on monosacchar$des and oligosaccharides. Such reactions, e.g. using periodate oxidation, are known. The reaction yields a product having two aldehyde groups for each sugar monomer in the oligosaccharide.
Thus, six aldehyde groups derive from a raffinose (o-raffinose) or maltotriose (o-maltotriose), for example. Within a homologous series, the number of molar equivalents of the polyaldehyde cross linkers, required to achieve the same extent of stabilization of the sub-units as a divalent aldehyde such as glucose, is inversely proportional to the valency of the CSSC. In contrast to GA, o-raffinose is intramolecular cross-linking specific, so that higher molar ratios of o-raffinose to Hb ~20:1) can be used in the preparation of polymer-free Hb(T). This discovery demonstrates that CSSC effectively stabilizes Hb monomers into specific conformation with minimal intermolecular cross-linking.
This affords more effective control of the reaction through slowing of the polymerization process, and allows easier production of an essentially polymer-free stabilized Hb product.
Specific examples of useful CSSC compounds include a homologous series of periodate oxidized saccharides of glucose, inulin, maltose, maltotriose, maltotetrose, maltopentose, maltohexose and maltoheptose. In general, the CSSC compounds derived from ring opening oxidation of sugars corraspond to the formula 71 l2 CHO CHO

or 1 l ~ 2 1 3 14 R - I - O ~ I - - Q - Cm H2 m - C - O - C - Rs CHO CHO CHO C~O

where each of R, Rl, R2, R3, R4 and R5 is independently selected from hydrogen, hydroxyl, methoxy and hydroxymethyl:
m is zero or one:
and Q ls a chemical group resulting from the oxidative ring opening of a di- or oligosaccharide having up to 36 sugar moieties.

When the starting compound is raffinose (a trisaccharide), the CSSC oxidation product has the fcrmula:

l l H H H
HC - C - O - IC - O - CH2 - C - O - f - O - C - O - CH - CH2OH
CHO H CHO CHO CHO CHO CHO

.

.. . . .

- 14a - 1306~83 The cross-ll~kins reagents used in the present in~entlon are thus quite dif erent from the DP5 analosues conventionally used in the prior art to stabilize and c~css-link Hb in its tetrameric form. They have no negatively charged groups such as phosphate, carboxylate or the like, and are not known specif$cally to reac_ with the DPG site of the Hb. Hence, they are able to effect the conformational stabi~ization of the Hb, of human and domestic animal origin, to retain each Hb unity in the conformation in which lt encounters and reacts with the cross-linker. Water soluble cross-linkers are preferred for ease of reaction.
The relatlve amounts of aldehyde to hemoglobin are preferably arranged to provide a molar rat~o of aldehyde groups to hemoglobin of at least 12:1, and preferably at least 60:1.
Thus, when polyaldehydes are used, less polyaldehyde is required for the same amount of hemoglobin, than in the case of , l~V~5~33 dialdehydes. The calculation of molar ratio is done on the assumption that hemoglobin has about 50 primary amine groups.
Since the globin chain linking reactions in the present invention do not need to be site specifio, excess molar ratio of aldehyde (e.g. for o-raffinos~, molar ratio of 5-30 can be used preferably 20:1) can be used so as to obtain high yields (at least 95~) of stabilized product. Prior art processes requiring site specific reaction must use substantially stoichiometric amounts of reagents, with consequent decreases in yield. Reactions suitably take place in aqueous solution, at temperatures ranging from 4 ~
37C preferably at ambient temperatures. The pH of the reaction mixture should be controlled to between 6.7 and 9.5, preferably 8.0, by suitable buffering.
When using GA as the cross-linking reagent, to prepare poly Hb(T), a lower conaentration of Hb in solution less than about 5-7 g/dl, should be used in order to exercise tha necessary control over the reaction. With the other CSSC products, however, a greater range of Hb concentration can be used, e.g. from 1-20 g/dl, preferably 3-4 g/dl, since the reaction is more easily controllable.
Stroma free hemoglobin as obtained from red blood cells has a hemoglobin concentration of about 3-4 g/dl. It is most convenient to use this hemoglobin solution without dilution or concentration thereof. The preferred concentrations depend upon the choice of aldehyde. Thus, advantages of the novel CSSC over divalent GA are applicable equally to the production of polymer-- 16 _ ~3Q6583 free human Hb derivatives and of polymer-free animal Hb d~rivatives, but the prevention of poly Hb formation, using the novel CSSC such as o-raffinose, permits use of animal-Hb-derived HemoSafe products ln man.
Stabilized Hemoglobin:
In preparation of the conformationally stabilized and cross-linked deoxy-Hb (XL-Hb(T)) of the invention, the intramolscular cross-linking reaction can be terminated prior to the onset of the intermolecular cross-linking to form dimers, trimers, tetramers and higher polymers, by addition of reducing or quenching agent at the appropriate time. The reaction of the primary amine gxoups with the aldehydic groups of the CSSC forms Schiff base linkages. Addition of sodium or potassium borohydride or the like reduces the Schiff base linkages to secondary amine linkages, and also reduces excess aldehydic groups to primary alcohols, thus arresting further cross-linking.
The appropriate time for reduction can be ascertained through high pressure liquid chromatography (HPLC) analysis of a sample of the reaction mixture.
The conformationally stabilized tetrameric Hb product may be recovered and purified by standard techniques. Thus, it may be washed and concentrated by membran0 filtration, and made up in physiological salt solution for use as a blood substitute, on an emergency basis where its rslatively short half-life in the vascular system is acceptable. Alternatively, it may be used as tha starting material for preparing other products according to the invention.
Polymerized stabilized hemoglobin One of such other products is polymerized Hb (HemoSafe II), in which the conformationally stabilized and dissociatively stabilized tetrameric hemoglobin ~HemoSafe I) is intermolecularly linked to form polymerized Hb, with a molecular weight preferably giving iso-oncotic pressure at a hemoglobin concentration of 14 g/dl. Polymerization is appropriately accomplished by use of dialdehydes or polyaldehydes, whlch may be the same as or different from those used for conferring conformational stability. Poly (Hb) products prepared in the prior art, for example, using glutaraldehyde have lacked the essentially complete conformational stability of the products of the present inventlon, since they have not been made from HemoSafe I.
Polymeric products according to the inventlon may contain a mixture of polymerlc species, differing from one another in respect of their molecular weight (number of units in the polymer). However, all the species are conformationally stabilized in the T-conformation. All the species have the same or substantially the same oxygen affinity. Accordingly, the polymerized products of the invention have the advantages over the prior art of homogeneous oxygen affinity and readily reproducible composition.
Suitable conditions of reaction to produce the polymers are aqueous solution at room temperature, and in any event not 13~6~33 exceeding about 37C. The ratio of aldehyde to hemoglobin is suitably 2 to 100 on the basis of moles of aldehyde group per mole of Hb. The reaction solution should be buffered to within a pH of 6.7 - 9.5, preferably 8Ø The reaction can be terminated by addition of reducing agent such as sodium borohydride or sodium cyanoborohydride thereto, to reduce the residual aldehyde groups, and to convert the Schiff base to stable covalent carbon-nitrogen linkage.
A particularly preferred polymeric product composition of the invention has a molecular weight distribution in which from 20 - 25% of the constituent polymers consist of five or more tetramerlc hemoglobin monomeric units, 50 - 60~ o~ the constituent polymers have 2 - 4 hemoglobin monomeric units, 20 -25~ of the constituents are a single hemoglobin unit (molecularwei~ht ~64 kilo dalton), and from 1 - 2% are incompletely stabilized hemoglobin. Such a composition is iso-oncotic at 14 g/dl, so that it can be used in equal volumes with blood as a blood substitute without causing blood pressure problems.
Stabilized-Hemoglobin con~ugates Another of such products is "conjugated HemoSafe I", in which the conformationally stabilized and dissociatively stabilized hemoglobin (HemoSafe I) is covalently conjugated to a bio-macromolecule to form a product of desirably high molecular weight. Conjugates of hemoglobin and bio-polymers (inulin, dextran, hydroxyethyl starch, etc.) are known in the prior art.
In the present invention, howevsr, whilst the same bio-polymers ~3~65~33 and similar methods of linking to hemoglobin can be adopted as in the prior art, the hemoglobin retains its conformational stability, preferably T, in the con~ugate form, with the attendant advantages discussed above.
Particularly preferred as the polymer for preparing conjugates according to the present invention is inulin. This is a polysaccharida of approximately 5000 molecular weight, known for use in diagnostic aids.
Pasteurized Product The prior art stabilization, polymerization, and conjugation processes described, do not protect the heme from oxidation, or the macromolecule from denaturation, when the macromolecule is sub~ected to heat, to drying or to freezing. It has now been discovered according to this invention, that the heme-containing macromolecule, when first stabilized, and optionally then polymeri~ed or conJugated as described above, can be protected from thermal oxidation, e.g. by use of oxygen displacers or scavengers, or preferably by formation of heme-protecting complexes 5uch as those formed by reaction with carbon monoxide (CO), or nitric oxide in such a manner as to form, for example, a carbon monoxy-heme derivative which permits pasteurization and freeze drying without denaturation.
Specifically, addition of CO to the novel, stabilized hemoglobins derived using GA, or using the CSSC products, yields . ~
novel products which are sometimes called herein CO-HemoSafe(s).

For this purpose, C~ may be added to the reaction mixture without 13~65~3 isolation of the hemoglobin product therefrom. In the already stabilized hemoglobin, the protection of the heme by C0, and prevention of allosterlc conformation changes together prevent oxidation and denaturation of C0-HemoSafe(s) under conditions of heat and freeze-drying. The carbon monoxylation and pasteurization as applied to HemoSafe I, HemoSafe II, and HemoSafe I - Inulin, or any other hemoglobin-based blood substitute, has not been described in the prior art.
Pasteurization (e.g. wet-heat at 60C for 10 hours) and lyophilization (freeze drying), of hemoglobin derivatives without addition of suitable protective agents leads to their precipitation and denaturation. By protecting the heme of the uniquely-stabilized hemoglobins of this invention with C0, heating and freeze drying of the product C0-HemoSafe can be successfully achleved. Successful pasteurization yields a soluble, undenatured C0-HemoSafe whlch is vlrus-dlsease transmission-free. Successful lyophilization then yields a dry C0-HemoSafe product. Both liquld and dry C0-HemoSafe products are stable to long term storage at any reasonable temperature, (e.g. 56C for 60 days). C0-HemoSafe is stable but does not dellver oxygen. Therefore, for use ln transfusion, C0-HemoSafe solution is converted to oxy-Hemosafe solution by treatment with light ln the presence of oxygen. Thus, another feature of this invention is the photoconversion of C0-HemoSafe to oxy-HemoSafe prior to transfusion.

13Q~5~3 The GA concentration (i.e. 5 mM) used in the inltial stages of the present process is greater than that of 1 mM reported in the prior art to be necessary to inactivate 95~ of the virus known as HTLV-III/LAV or HIV-I. However, assurance of viral transmission safety could be achieved by pasteurization, that is, heating to 60C for ten hours, which is the accepted standard treatmsnt for plasma-derived pharmaceutical products like albumin. If, for example stabilized oxy-Hb(T), derived as above, were sub~ected to 60C for ten hours, a brown precipitate ~i.e. denatured met-Hb(T)) would form, which is unsuitable for further use. However, through conversion of stabilized Hb(T) to its carbonmonoxy derivative, C0-HemoSafe I(T) is formed which tolerates pasteurization and storage for up to 60 days in solution at 25C or 56~C, with no detectable change n physical-chemical properties such as its visible spectrum, oxygen affinity (i.e. P50 ), or composition, as indicated by its HPLC profile. In addition, C0-HemoSafe I(T) can be freeze-dried and stored at 25C
or 56~C for 60 days without detectable changes in physical-chemlcal properties as described for pasteurization above.
Transfusible product Pure C0-HemoSafe does not bind or transport oxygen, therefore it is unsuitable for oxygen delivery and transfusion despite its stability throughout the preparation, pasteurization and freeze drying procedures. Removal of CO under aseptic conditions is a prerequisite for its use as a blood substitute.
The present invention, therefore, teaches that C0-HemoSafe can be readily photoconverted to the oxygenated derivative oxy-Hemoglobin. For this, oxygen may be supplied to the solution, and the solution may then be passed through a transparent tubing exposed to visible llght. Optical absorpt~on spectra of CO-HemoSafe and oxy-HemoSafe provide a convenient means of characterizing and quantitating the two species. In contrast to existing blood substitutes, like the currently available poly PLP-Hb, HemoSafe has the advantage of physiological and uniform oxygen affinity Its precursor, CO-HemoSafe, displays stability under conditions of pasteurization, lyophilization, photoconversion and storage at any temperature up to 60C. Thus, this invention teaches processes which produce a sterile viral-disease transmission-free and efflcacious blood substitute.
In addition, in accordance with this inventlon, it has been found that the stability conferred by the process of carbon monoxylation can be applied to any Hb-based blood substitute described in prior art. For example, it has been demonstrated, according to the invention, that carbonmonoxy derivatives of poly PLP-Hb (poly CO-PLP-Hb), of ATP-Hb (CO-ATP-Hb), and of poly ATP-Hb (poly CO-ATP-Hb), are equally stable, pasteurizable, freeze dryable and photoconvertible to their oxy derivatives. Thus, the present invention includes processes applicable as a general requisite treatment to eradicate pathogenic viruses from oxygen carrying resuscitation fluids based on hemoglobin, and to improve their storage properties. Hence, all existing hemoglobin based blood substitutes such as dextran-Hb, hydroxyethyl starch Hb, 1306i~j~33 polyethylene glycol-Hb, and diaspirin cross-linked Hb can be rendered virus-disease transmission free by the same processes described in the present application.
The preferred practical production process of HemoSafe of the present invention may be summarized as follows:
(i) Pooled whole blood is diluted with isotonic NaC1 solution and applied to a membrane-type filtration system.
(ii) Plasma is separated from cellular elements of blood by tangential flow membrane filtration.
(iii) Red blood cells are washed in isotonic saline.
(iv) Red blood cells are lysed with hypotonic phosphate buffer. Then cell debris and other particulate matter are separated from soluble hemoglobin by tangential flow filtration.
(v) Oxy-Hb ls converted to deoxy-Hb by vacuum or gas exchanging deviae and a reducing agent.
(vi~ Deoxy-Hb is stabllized by the addltion of a conformation specific stabilizing cross-linker (CSSC), in the monomer or polymer composition or con~ugate as desired.
(vii) Deoxy-Hb is physically stabilized with carbon monoxide, yielding CO-HemoSafe.
(viii) CO-HemoSafe is washed and concentrated, or electrolyte balance is reestablished by addition of appropriate physiological salt solutions.
(ix) Pasteurization proceeds on the CO-HemoSafe.

- 24 ~ 6S~3 x ) Pasteurlzed C0-HemoSafe is photoconverted, in the pres~nc~ of oxy~en, to oxy - H~moSafe.
(xi) Oxy-HemoSafe iS stsrile filt~red and packaged for transfusion.

Once oxy-HemoSaf~ b~comes available, as taught by this invention, it is possible to convert it to thè met-hemoSafe derivative for use as an antidote and as a preventive tran~fusion in anticipation of cyanide poisoning such as in chemical defence.
Thus, this invention teaches that after preparat~on of oxy-HemoSafe, known methods are used to oxidize it into met-HemoSafe, which can be transfused as an instantly effectiva and long acting cyanide scavenger. Using met-HemoSafe intravenously avoids the time delay attending of the body's utilization of oral nitrites to form met-Hb. Nltrites also necassarily recruit met-Hb from exlsting RsC oxyhemoglobln, thus use of met-HemoSafe does not compromise existing oxygen carrying capacity. Therefore, met-HemoSafe provides a treatment for cyanide intoxication beyond that used in prior art.
Transfusion of oxy-hemoSafe also can be used as a substitute for the blood doping techni~ue. Whereas prior art teaches that red blood cells can be given to individuals to enhance their oxygen carrying capacity, the viscosity attending the red cells limits this increase to 10-15~. However, by using oxy-HemoSafe in conjunction with the known plasmapheresis technique, plasma can be replaced with oxy-HemoSafe, theoretically, increasing the 13~iS8~

oxygen carrying capacity by 50%, wi-thout changing the hematocrit or flow characteristics of the blood.
Whilst it is not intended that the inventio~ or its scope should be limited to any particular theory of the structure of the products or mechanisms of the processes for their preparation, it is believed probable that the conformational stability and dissociative stability of the products may derive from intra~subunit, covalent linkages in the tetrameric hemoglobin by CSSC. These linkagas are stable enough to prevent dissociation into dimers, and rigid enough to prevent conformational change. Whatever the structural explanation, the HemoSafes are unique in their conformational stability.
Moreover, the present invention is not limited in its scope to hemoglobin treatments and hemoglobin products. It is applicable to other proteins capable of existing in one or more conformations. Treatment of such proteins, whether in solution or in biological membranss with a CSSC compound according to tha invention will stabilize the conformation in which the proteins encounter the CSSC. In theory, this should also stabilize biological activity associated with the stabilized conformation and render the molecule stable to pasteurization. Thus, it is of wide applicability to biological macromolecules including biologically active peptides, many of which are useful only in a particular conformation.
The invention is further described in the following 1306~

specific, non-limiting examples, to which the various figures of drawings apply, as follows:

In the accom~anying dra~in~s:
FIGUR~ 1 is the analytical gel perm~ation High Pre~sure Liquid Chromatography (HPLC) profile of the stabilized human deoxy hemoglobin prod~ced according to ~xample 3 below, with a comparison to SFH;

FI~UR~ 2 is an oxy~en dissociation curve of the HemoSafe (1) products of Examples 3 and 4;

FIGURE 3 shows absorptlon SpeCtra Of the products of Example 3 and Example 15:

FIGURE 4 is a graphlcal presentation of half-life studles of the products of Example 3, Example 9 and SFH;

FIGURE 5 is the analytical gel permeation High Pressure Liquid Chromatography (HPLC) profile of products according to the procedure of Example 9.

Example 1 PREPARATION OF HUMAN AND ANIMAL STROMA-FREE HEMOGLOBIN
Human stroma-free hemoglobin was prepared from outdated blood or packed blood cells obtained from the Canadian Red Cross.

13~ 3 First, the whole blood was centrifuged at 3,000 rpm for 30 minutes. Then, the plasma and buffy coat were removed by suction through a pipette and discarded. The sedimented erythrocytes were washed three times by suspendin~ them in 3 times their volume of ice-cold normal saline. Following each wash, the cells were re-sedimented by centrifugation and the supernatant removed and discarded.
Next, the washed red cells were lysed with 4 volumes of 5 mM
phosphate buffer, pH 7.6, to rupture the intact cell wall and free the hemoglobin. To remove stroma and membrane fragments, the hemolysate was filtered by a Pellicon Cassette System (Millipore) with a fluorocarbon polymer filter (HVLP, Porosity 0.5 ,um), followed by a polysulphone filter (100,000 molecular weight cut-off). The hemoglobin in the filtrate was first concentrated to 14-20 g/dl and then washed with 10 volume excess l of PBS buffer, pH 7.4, with a Pellicon Cassette System equlpped with 5.0 sq.ft. of membrane cassette (PT series, 30,000 molecular weight cut off) and sterilized by filtration through a 0.22 ,um Millipore filter unit and stored at 4C until use.
For animal hemoglobin preparations, the same procedure as that of human hemoglobin was used with the exception of rat hemoglobin where a 20 mM Borate buffer (pH 9.5) was substituted for the PBS buffer (pH 7.4) to enhance solubilization of the hemoglobin.

~r ~

13Q65~3 - 2~ -Example 2 PREPARATION OF PERIODATE OXIDIZED RAFFINOSE AND OTHER SU~ARS AS
CSSC FOR HEMOGLOBIN
200 mg each of glucose, sucrose, raffinose, maltose, maltotriose, maltopentose, maltohexose, maltoheptose and other sugars in 6 ml of distilled H2O were treated with a fixed molar ratio of solid sodium m-periodate per saccharide at room temperature. (The molar ratio of sodium m-periodate per saccharide used was 2.2 for pyranoside and 1.1 for furanoside).
After one hour, the reaction mixture was cooled in an ice-water bath, sodium bisulfite was added with vigorous stirring until the precipitated iodine was redissolved to yield a colorless solution. The pH of the solution was then immediately adjusted to 8.0+ 0.1 by the addition of 6N NaOH. The resulting oxidized sugar solutions were further diluted to give a final concentration of 20 mg/ml and filtered through a Milllpore 0.45 ,um type GS membrane filter and stored at 4C until use.

Example 3 PREPARATION OF HUMAN HEMOSAFE I (T) CONFORMATIONALLY STABILIZED
AND CROSS-LINKED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of lntramolecularly cross-linked deoxyhemoglobin (XL-Hb(T)): A 350 ml solution of stroma free oxyhemoglobin (Hb(R)) 1% W/V in 0.1 M phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin (Hb~T)) under vacuum for approximately 4 hours at room temperature. 0.1 m mole of sodium dithionite dissolved in 0.3 ml of degassed buffer was - 29 _ 13~65~3 added to the hemoglobin solution and allowed to react for 5 minutes. The cross-linkin~ agent, o-raffinose, 1.08 m mole in 20 ml of previously degassed buffer, pH 8.0, was added under vacuum with vigorous stirring and the solution stirred for 4 hours at room temperature. The reaction mixture was cooled in an ice-water bath, and 15.0 m moles of sodium borohydride in 5 ml of degassed 1 mM NaOH under positive inert gas pressure was added.
Reduction was allowed to proceed for 45 minutes.
Figure 1 is the HPLC profile obtained from 5 ~g/100 ml of the product (solid line), and a control human SFH (dashed llne). Molecular weight calibrations indicative of B-globin half molecules (peak a) and (~ B)2 stabilized human Hb(T), (peak b) are as marked. The buffer used was 50 m M phosphate and 150 m M NaCl, pH 7Ø The chromatogram was obtained on a Pharmacia FPLC system equipped with GP.250 gradient programmer, P500 pump, a model 482 chart recorder and a single path UV monitor with 405 nm filter.
The profile shows that greater than 95% of the hemoglobln is stabilized and cross-linked and less than 5% of the XL-Hb(T) dissoclate lnto half-molecules. Under the same experimental conditions, hemoglobin completely dissociates into half-molecules.
Preparatlon of the carbon monoxide (CO) derivative of XL-Hb(T) (COXL-Hb(T)) and pasteurization: Following the reduction with sodium borohydride, CO was bubbled directly lnto the XL-Hb(T) reaction mlxture. After washing and concentration by membrane flltration, the COXLHb(T) under 100~ CO was pasteurized ~31~6583 at atmospheric pressure by heating for 10 hours at 60C to give C0-HemoSafe I. For long term storage at ambient temperature, cO-HemoSafe I can be lyophilized to a dry powder and reconstituted with a buffer when needed.
Photoconversion of C0-HemoSafe I ( T ) to oxy-HemoSaf~ I ( T ):
15 ml of C0-HemoSafe I(T) was transferred into a 500 ml round bottom flask which was attached onto a rotary evaporator and continuously rotated in an ice-water bath at O~C under a CGE
Brooder Lamp. (C 250 R40/1, inside frost, soft glass) The flask was evacuated with an aspirator or a vacuum pump for the removal of unbound C0. After 2 minutes, air or 100~ oxygen was introduced. This cycle was repeated until the composition of the sample checked spectrophotometrically between 577 and 560 mm gave an absorbency ratio of 1.8, after which removal of C0 was considered complete (Methods in Enzymology, vol. 76, Hemoglobin, pp. 60 & 164). The final product so obtained has unaltered gel permeatlon chromatographic composition, shown in Figure 1.
Flgure 3 illustrates the relative absorption spectra of the products of thlS and other axamples, in Ringer's buffer pH
7.4 at 22C. The solid line i5 that of the XL-Hb(T), i.e. prior to carbon monoxylation, and the dotted line is that of the oxy-HemoSafe final product. These curves on Figure 3 show that the final product has essentially unaltered physical-chemical properties.
Figure 2 is the oxygen dissociation curve o~ the HemoSafe (1) cross-llnked and stabilized by o-raffinose in this Example (dashed line) and that of oxy-hemoglobin or Hb(R) (solid 13~6583 ~ 31 -line), measured at 37C in Ringer's phosphate buffer at pH 7.4 with hemoglobin concentration at 3.5 g/dl. These are typical curves of such products. Values of partial pressure of oxygen at 50~ saturation (Pso) are read directly from the curve.
The HemoSafe (1) product of this Example shows according to Figure 2 a hyperbolic oxygen dissociation curve with a P5 o value (Pso is partial pressure of oxygen at which 50% of the hemoglobin is in the oxygenated state) of 27 mmHg at 37 D C in PBS
buffer~ (pH 7.4). It has a Hill coefficient of -1-1.5.
On Figure 4, there are presented typical plasma half-life studies of hemoglobin-based blood substltutes including, on the dashed line, that of the final oxy-HemoSafe l(T) product of this Example. The measurements are made in rats in a 30%
hypervolemic model. The product has a half-life of approximately 4-5 hours as compared with that of hemoglobin (dotted line) of about 1-1.5 hours.

Example 4 PREPARATION OF HUMAN HEMOSAFE I(R) CONFORMATIONALLY STABILI~ED
AND CROSS-LINKED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) The procedure is similar to that of HemoSafe I(T), described in Example 3, with the excaption that the reaction was carried out under oxy state and in the absence of sodium dithionite.
The final product shows a hyperbolic oxygen dissociation curve with P50 value of 2-3 mmHg at 37C in PBS buffer, pH 7.4, (Figure 2, solid line) and its gel permeation chromatographic ~3C~ 3 composition and visible spectrum are indistinguishable from those of HemoSafe I(T).

_ample 5 PREPARATION OF HUMAN HEMOSAFE I(T) CONFORMATIONALLY STABILIZED
AND CROSS-LINKED BY GLYOXAL
Preparation of intramolecularly cross-linked stroma-free deoxyhemoglobin with glyoxal: Human stroma-free hemoglobin 2.9 g, 4% w/v in O.lM sodium bicarbonate under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 2 hours. The cross-linking agent, glyoxal, 2.58 m moles in 7.5 ml of degassed O.l M sodium bicarbonate was added and the solution stirred for 2 hours at room temperature. The reaction mixture was cooled in an ice-water bath and 6 m moles of sodium borohydide in 3 ml of degassed 1 mM NaOH was added under positivs inert gas pressure. Reduction was allowed to proceed for 45 minutes. The HPLC gel permeation profile is similar to that of Figure 1 which shows that greater than 90% of the hemoglobin is intramolecularly stabilized.
The procedures for the preparation of glyoxal stabilized CO-HemoSafe I ( T ), its pasteurization, lyophilization and photoconversion back to oxy-HemoSafe I ( T ) are similar to those described in Example 3. The final product has a half-life of about 3.5 hours measured in tha rat with a 30~ hypervolemic replacement.

1~0~3 Example 6 PREPARATION OF BOVINE HEMOSAFE I(T) CONFORMATIONAL~Y STABILIZED
AND CROSS-LINKED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of intramolecularly cross-linked bovine deoxyhemoglobin: A 90 ml solution of bovine stroma-free oxyhemoglobin, 1% w/v in O.lM phosphate buffer pH 8.0 under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 2 hours at room temperature. 400 ~ moles of sodium dithionite dissolved in 0.3 ml of degassed buffer was added to the hemoglobin solution and allowed to react for 5 minutes. The cross-linking agent, o-raffinose, 276 ~ moles in 6 ml of previously degassed buffer, pH 8.0 was added under vacuum and the solution stirred for 3 hours at room temperature. The reaction mlxture was cooled in an ice-water bath, and 3.5 m moles of sodium borohydide in 2 ml of 1 mM NaOH (degassed) was added under positive inert gas pressure. Reduction was allowed to proceed for 45 minutes. The HPLC gel permeation chromatograpic proflle shows that >90% of the bovine hemoglobln was intramolecularly stabilized. The chromatogram of cross-llnked bovine hemoglobin ls simllar to that shown in Figure 1.
The procedures for the preparation of o-raffinose stabilized bovine CO-HemoSafe I(T), its pasteurization, lyophilization and photoconversion to oxy-Hemosafe I(T) are similar to those desired in Example 3.
The final product so obtained has unaltered gel permeation chromatographic composition and physical-chemical properties.

- 34 - ~3~6~83 The product has a hyperbolic oxygen dissociation curve with P5 o of 34 mmHg measured at 37C in PBS buffer, pH 7.2, a Hill coefflcient of ~1.5 and a plasma half-life of ~4 hours measured in 30~ hypervolemic transfusion in rat.

E~ample 7 PREPARATION OF OVINE HEMOSAFE I(T) CONFORMATIONALLY STABILIZED
AND CROSS-LINKED BY RING-OPENED RAFFINOSE (O-RAFFINOSE).
Preparation of intramolecular cross-linked ovine deoxyhemoglobin: A 100 ml solution of sheep stroma-free oxyhemoglobin, 1% w/v in O.1 M phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 2 hours at room temperature. 50 ~ moles of sodium dithionite in 100 ,ul of degassed buffer was added to the hemoglobin solution. After 5 minutes, the cross-linking agent, o-raffinose, 616 ,u moles in 12.5 ml of previously degassed buffer pH 8.0 was added under vacuum with stirring. Cross-linking was allowed to proceed for 16 hours at 4~C. The reaction mixture was cooled in an ice-wate~ bath, and 4.4 m moles of sodium borohydride ln 2.5 ml of degassed 1 mM NaOH was added under positlve lnert gas pressure. Reductlon was allowed to proceed for 45 minutes. The HPLC gel permeation chromatographic profile revealed that greater than 95% of the ovine hemoglobin half-molecule was intramolecularly stabilized, similar to that shown in Figure 1.

13Q65~33 The procedures for the preparation of o-raffinose stabilized ovine CO-HemoSafe I(T), its pasteurization, lyophilization and photoconversion back to oxy~HemoSafe I ( T ) are similar to those described in Example 3.
The final product so obtained has unaltered gel permeation chromato~raphic composition and physical-chemical properties.
Th~ product has a P50 of 38 mm~g measured at 37C in PBS buffer, pH 7.4.

Example 8 PREPARATION OF RAT HEMOSAFE I(T) CONFORMATIONALLY STABILIZED AND
CROSS-LINKED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of intramolecular cross-linked rat deoxyhemoglobln: A 18 ml solution of rat stroma-free oxy hemoglobin, 2% w/v in 20 mM Borate buffer, pH 9.5, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 1 hour at room temperature. 50 ,u moles of sodium dithionite dissolved in 0.1 ml of degassed buffer was added to the hemoglobin solution and allowed to react for 5 minutes. The cross-linking agent, o-raffinose, 110 ,umoles in 3 ml of pxeviously degassed buffer, pH 8.0, was added under vacuum with stirring . Cross-linking was allowed to proceed for 16 hours at 4~C. The reaction mixture was cooled in an ice-water bath and 1.2 m moles of sodium borohydride in 1 ml of 1 mM NaOH was added under positive inert gas pressure. Reduction was allowed to proceed for 45 minutes. The HPLC gel permeation chromatographic - 36 - ~3065~3 profile shows that greater than 90% of the rat hemoglobin half-molecule was intramolecularly stabilized similar to that shown in Figure 1.
The procedures for the preparation of o-raffinose stabilized rat CO-HemoSafe I ( T ), its pasteurization, lyophilization, and photoconversion back to oxy-HemoSafe I(T) are similar to those described in Example 3.
The final product obtained has unaltered gel permeation chromatographic composition and physical-chemical properties.
The product has a P50 of 24 mmHg measured at 37C in PBS buffer, pH 7.4, and a ~ill coefficient of ~ 1Ø

Example 9 PREPARATION OF HUMAN HEMOSAFE II CONFORMATIONALLY STABILIZED AND
POLYMERIZED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of intramolecularly stabilized and intermolecularly cross-linked human deoxyhemoglobin: A 77 ml solution of human oxyhemoglobin, 4% w/v in 0.1 M phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 4 hours at room temperature. 0.10 m moles of sodium dithionlte dissolved in 0.3 ml of degassed buffer was added to the hemoglobin solution and allowed to react for 5 minutes. The cross-linking agent, o-raffinose, 0.95 m moles in 20 ml of previously degassed buffer was added under vacuum and the reaction allowed to proceed for 6 hours. The reaction mixture was cooled in an ice-water bath, and - 37 - ~3065~3 9.5 m moles of sodium borohydride in 4 ml of degassed 1 mM NaOH
was added under positive inert gas pressure. Reduction was allowed to proceed for 45 minutes. In accompanying Figure 5, the HPLC gel permeation chromatographic profile revealed that greater than 98~ of the hemoglobin was stabilized and cross-linked with 20-25~ being the stabilized monomer, 55 5~ being the dimers, trimers and tetramers combined and 20-25% being the pentamers and higher polymers combined.
This chromatographic profile was obtained using as buffer 50 mM phosphate and 150 mM NaCl, pH 7, with the apparatus described for Figure l. The poly Hb(T) was resolved into monomers (peak a), the sum of dimer, trimer and tetra~er (peak b) as well as pentamers or greater (peak c), on a Superose-12 prepacked HR 10/30 column at a flow-rate of 0.~ ml/min. Such a composition of the polymerized product was shown to be iso-oncotic at a concentratlon of 14 g/dl.
The procedures for the preparation of o-raffinose stabilized CO-HemoSafe II(T), its pasteurization, lyophilization and photoconversion back to oxyHemoSafe II(T) are similar to those described in Example 3.
The final product so obtained has unaltered gel permeation chromatographic compositlon and physical-chemlcal properties before and after pasteurization, has a hyperbolic oxygen dissociation curve with uniform P5 o of 32 mmHg, and a Hill coefficient 1.6 measured at 37C in PBS buffer, pH 7.2. The isolated purified fractions of HemoSafe II(T), i.e. monomer, ~3C~6S~33 dimer, trimer, tetramer, pentamer or greater (see Figure 5) has indistinguishable P5 o, a Hill coefficient and a half-lifa of 7-8 hours measured in rat with a 30~ hypervolemic replacement.

Example 10 PREPARATION OF HUMAN HEMOSAFE II CONFORMATIONALLY STABILIZED AND
POLYMERIZED BY RING-OPENED SUCROSE (O-SUCROSE) Preparation of intramolecular stabilized and intermolecular cross~linked human deoxyhemoglobin by o-sucrose: A 10 ml solution of human stroma-free oxyhemoglobin, 13~ w/v in 0.1 M
phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 2 hours. 40 ,u moles of sodium dithionite dissolved in 0.2 ml of degassed buffer was added to the hemoglobin solution. After 5 minutes, the cross-linking agent, o-sucrose, 200 ,u moles in 3 ml of previously degassed buffer was added under vacuum and the reactlon allowed to proceed for 6 hours. The reaction mixture was cooled in an ice-water bath, and 2.0 m moles of sodium borohydride in 1 ml of degassed 1 mM NaOH was added under positive inert gas pressure.
Reduction was allowed to proceed for 45 minutes~ The HPLC gel permeation chromatographic profile revealed that >98~ of the hemoglobin was stabilized and cross-linked and the product composition is similar to that described in Example 9. ~Figure 5) The procedures for the preparation of o-sucrose stabilized CO-HemoSafe II(T), its pasteurization, lyophilization and _ 39 _ ~30~5~3 photoconversion back to oxyH~moSafe II ( T ) are similar to those described in ~xample 3.
The final product so obtained has unaltered yel permeation chromatographic composition and phys:Lcal-chemical properties before and after pasteurization. The product has a P50 of 24 mmHg msasured at 37C in PBS buffer, pH 7.2, a Hill coefficient of 1.3 and a half-life of ~7-8 hours in rat, measured at 30 hypervolemic replacement.

Exa~ple 11 PREPARATION OF HUMAN HEMOSAFE II(T) CONFORMATONALLY STABILIZED
AND POLYMERIZED BY GLUTARALDEHYDE
Preparation of intramolecular stabilized and intermolecular cross-linked human deoxyhemoglobin by glutaraldehyde: A 78 ml solution of human stroma-free oxyhemoglobin, 4% w/v in 0.1 M
phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 4 hours at room temperature. 0.1 m moles of sodium dithionite dissolved in 0.3 ml of degassed bu~fer was added to the hemoglobin solution and allowed to react for 5 minutes. The cross-linking agent, glutaraldehyde, 480 ~ moles in 2.5 ml of previously degassed buffer was added under vacuum and the reaction allow to proceed for 6 hours. The reaction mixture was cooled in an ice-water bath and 4 m moles of sodium borohydride in 4 ml of degassed 1 mM
NaOH was added under positive inert gas pressure. Reduction was allowed to proceed for 45 minutes. The HPLC gel permeation ~3~?65~33 chromatographic profile shows that >98% of the human hemoglobin was stabilized and cross-linked and the product composition is similar to that described in ~xample 9.
The procedures for the preparation of glutaraldehyde stabilized CO-HemoSafe II ( T ), its pasteurization, lyophilization and photoconversion back to oxy-HemoSafe II(T) are similar to those described in Example 3.
The final product so obtained has unaltered gel permeation chromatographic composition and physical properties. The product has a P50 f ~30 mmHg measured at 37C in Pss buffer pH 7.4 and a Hill coefficient of 1.5 and a half-life of 7-8 hours, measured in rat with 30% hypervolemic replacement.

Example 12 PREPARATION OF BOVINE HEMOSAFE II(T) CONFORMATIONALLY STABILIZED
AND POLYMERIZED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of intramolecular stabilized and intermolecular cross-linked bovlne deoxyhemoglobin by o-raffinose: 40 ml solution of bovine stroma-free oxyhemoglobin, 6~ w/v in 0.1 M
phosphate buffer, pH 8.0, under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 4 hours at room temperature. 80 ,u moles of sodium dlthlonite dissolved in 0.2 ml of degassed buffer was added to the hemoglobln solution and was allowed to react for 5 minutes. The cross-linking agent, o-raffinose, 550 ~ moles in 10 ml of previously degassed buffer was added under vacuum and the reaction allowed to proceed for 6 - 41 _ 13~S~3 hours. The reaction mixture was cooled in an ice-water bath and 3.5 m molss of sodium borohydride in 4.0 ml of degassed 1 mM NaOH
was added under positive inert gas pressure. Reductlon was allowed to proceed for 45 minutes. The HPLC gel permeation chromatographic profile shows that >95~ of the bovine hemoglobin half-molecule was stabilized and cross-linked and the product composition is similar to that described in Example 9.
The procedures for the preparation of o-raffinose stabilized bovine CO-HemoSafe II(T), its pasteurization, lyophilization and photoconversion back to oxy-HemoSafe II(T) are similar to those described in Rxample 3.
The final product so obtained has unaltered gel permeation chromatographic composition and physical properties before and after pasteurization. The product has a P5 ~ of 28 mmHg measured at 37C in PBS buffer, pH 7.4, and a Hlll coefficient of ~1.5 and a half-life of 7-8 hours measured in rat with a 30% hypervolemic replacement.

Example 13 PREPARATION OF RAT HEMOSAFE II(T) CONFORMATIONALLY STABILIZED AND
POLYMERIZED BY RING-OPENED RAFFINOSE (O-RAFFINOSE) Preparation of intramolecular stabilized and intermolecular cross-linked rat deoxyhemoglobin: A 6 ml solution of rat stroma-free oxyhemoglobin, 9.4% w/v in 20 mM Borate buffer pH 9.5 under magnetic stirring was converted to deoxyhemoglobin under vacuum for approximately 2 hours at room temperature. 20 ,u moles of l3n6ss3 sodium dithionite dissolved in 100 ~1 of degassed buffer was added to the hemoglobin solution and allowed to react for 5 mimltes. The cross-linking agent, o-raffinose, 87 ~ moles in 2 ml of previously degassed buffer was added under vacuum and the reaction allowed to proceed for 16 hours at 4C, then 5 hours at room t~mperature. The reaction mixture was cooled in ice-water bath and 1.2 m moles of sodium borohydride in 1.0 ml of degassed 1 mM NaOH was added under positive inert gas pressure. Reduction was allowed to proceed for 45 minutas. The HPLC gel permeation chromatographic profile shows that >98~ of the rat hemoglobin half-molecule was stabilized ~nd cross-linked and the product composition ls Qimilar to that described ln Example 9.
The procedures for the preparation of o-raffinose stabilized rat CO-HemoSafa II(T), its pasteurization, lyophilization and photoconversion back to oxy-HemoSafe IItT) are similar to those descrlbed in Example 3.
The final product so obtained has unaltered gel permeation chromatographic composition and physical-chemical properties before and after pasteurization.

Example 14 PREPARATION OF RING-OPENED RAFFINOSE CONFORMATIONALLY STABILIZED
HUMAN HEMOSAFE I(T) - INULIN CONJUGATE
Preparation of intramolecularly cross-linked human HemoSafe I ( T ) . The preparation is identical to that described in Example 3.

1~65~3 Preparation of periodate oxidized Inulin (o-Inulin): The preparation of ring-opened inulin is similar to that described in Example 2.
Preparation of HemoSafa I(T) - Inulin conjugate: A 35 ml solution of human oxy-HemoSafe I ( T ), 4% w/v in O .1 M NaHCO3 under magnetic stirring was converted to deoxyHemoSafe I(T) under vacuum for approxlmately 2 hours at room temperature. 40 moles of sodium dithionlte dissolved in 50 ,ul of degassed buffer was added. After 5 minutes, equimolar of o-Inulin in 8.5 ml of previously degassed O.1 M NaHCO3 was added under vacuum with stirring and the reaction allowed to proceed for 5 hours at room temperature. The reaction mixture was cooled in an ice-water bath, and 2.0 m moles of sodium borohydride in 1.5 ml of degassed 1 mM NaOH was added under positive inert gas pressure. Reduction was allowed to proceed ~or 45 minutes. The HPLC gel permeation chromatographic profile and product composition of HemoSafe I(T) - Inulin con~ugate is similar to that shown in Figure 5. The procedures for the preparation of raffinose stabilized human CO-HemoSafe I(T) ~ Inulin con~ugate, its pasteurization, lyophilization, and photoconversion back to human oxy-HemoSafe I(T) - Inulin conjugate are similar to those described in Example 3.
The final product so obtained has unaltered gel permeation chromatographic composition and physical-chemical properties.
The product has a P50 of 27 mm Hg at 37C in PBS buffer, pH 7.2, - 44 _ 130~3 a Hill coefficient of ~1.2, and a half-life of 7-8 hours measured in rat with a 30~ hypervolemic replacement.

Example 1_ PREPARATION OF O-RAFFINOSE CONFORMATIONALLY STABILIZED HUMAN
MET-HEMOSAFES I (T) AND (R) After photoconversion of the CO-HemoSafes I ( T ) ~nd ( R ) to their respective oxy-HamoSafes as described above, (Examples 3), the oxyHemoSafes were left at 37C to allow their conversion to met-HemoSafes. The conversion is considered complete aftar met-hemoglobin levels are >90% as characterized by the optical absorption spectra, presented as the dashed line on Figure 3.
Its efficacy as a CN scavenger is ascertained by itS ability to yield the corresponding cyanomet-hemoglobln optlcal absorption spectrum, presented as the dashed-dotted line on Figure 3.

Example 16 PREPARATION OF O-RAFFINOSE CONFORMATIONALLY STABILIZED AND
POLYMERIZED HUMAN MET-HEMOSAFE II (T) AND (R) O-raffinose conformationally stabilized and polymerized human HemoSafe II (T) and (R) were prepared according to Example 9. These were converted to their respective met-HemoSafes as described in Example 15.

_ 45 _ 13~65~3 Example 17 PREPARATION OF O-RAFFINOSE CONFORMATIONALLY STABILIZED HUMAN MET-HEMOSAFE I ( T )-INULIN CONJUGATE
O-raffinose conformationally st~bilized human HemoSafe I(T)-inulin conjugate was prepared according to Example 14 and converted to the met-HemoSafe as described in Example 15.

Exa~ple lS
VIRAL INACTIVATION OF RAFFINOSE CONFORMATIONALLY STABILIZED AND
POLYMERIZED HUMAN HEMOSAFE II
Human HemoSafe II was prepared accordiny to Example 9.
Samples were prepared consisting of HemoSafe II, HemoSafe II
spiked with 104 HIV units, and 104 HIV units only. In control experiments, it was demonstrated that the presence of HemoSafe II
in solution does not affect the infectlvity of HIV-I. These samples were subjected to wet-heat at 56C for one week. At the end of this treatment, the samples were assayed for HIV
infectivity using an antigen assay. No HIV infectivity was detected in any of the samples.

Claims (24)

1. A conformationally stabilized hemoglobin product consisting essentially of tetrameric hemoglobin units which are both stabilized against dissociation into dimeric hemoglobin units, and are also conformationally stabilized against conforma-tional change between the T-conformation and the R-conformation upon formation of aqueous preparations thereof, said tetrameric units having secondary amino covalent chemical linkages between globin chains of the sub-units so as to effect the stabilization, the linkages having been formed by reaction of the hemoglobin with a polyaldehyde to form Schiff base links, followed by reduction to secondary amino linkages.

2. Stabilized hemoglobin product according to claim 1 which is conformationally stabilized into the T-conformation.

3. Stabilized hemoglobin product according to claim 2 formed by reaction of the hemoglobin with a polyaldehyde produced by oxidative ring opening of an oligosaccharide having at least two sugar moieties.

4. Stabilized hemoglobin product according to claim 3 wherein the oligosaccharide has up to seven sugar moieties per molecule.

5. Stabilized hemoglobin product according to claim 4 wherein the oligosaccharide is raffinose.

6. Stabilized hemoglobin product according to claim 1, claim 2, claim 3, claim 4 or claim 5, wherein the hemoglobin is of human origin.

7. Stabilized hemoglobin product according to claim 1, claim 2, claim 3, claim 4 or claim 5, wherein the hemoglobin is of animal origin.

8. Stabilized hemoglobin product comprising a plurality of stabilized tetrameric hemoglobin units according to claim 1, covalently linked together to form a polymeric hemoglobin product in which each hemoglobin unit has conformational stability and dissociative stability.

9. A stabilized polymeric hemoglobin product according to claim 8 wherein each hemoglobin unit thereof is stabilized in the T-conformation.

10. A stabilized polymeric hemoglobin product according to claim 9 and having a composition such that from 20 - 25% of the constituents are hemoglobin tetrameric units, 50 - 60% of the constituents are hemoglobin oligomers of 2 - 5 tetrameric hemoglobin units, 20 - 25% of the constituents are single hemoglobin tetramers, and 1 - 2% are dimers of hemoglobin subunits.

11. A process for preparing an aqueous solution of tetrameric, conformationally stabilized hemoglobin, which comprises reacting hemoglobin, in aqueous solution, with a polyaldehyde crosslinking reagent to form covalent Schiff base linkages between chains of the same tetrameric hemoglobin unit followed by reduction of said linkages to secondary amino linkages, and obtaining from the reaction mixture an aqueous solution of hemoglobin in which at least 95% of the hemoglobin is in the conformationally stabilized, tetrameric form.

12. The process of claim 11 wherein the polyaldehyde crosslinking reagent has at least four aldehydic groups per molecule.

13. The process of claim 12 wherein the crosslinking reaction takes place at a molar ratio of aldehyde groups on the crosslinking reagent to hemoglobin of at least 12:1, and at a temperature in the approximate range 4° - 37°C.

14. The process of claim 13 wherein the crosslinking reaction also takes place at a hemoglobin concentration in aqueous solution of from about 1 - 20 g/di, and a pH of from 6.7 - 9.5.

15. The process of claim 14 wherein the polyaldehyde is the product of ring-opening oxidation of an oligosaccharide.

16. The process of claim 15 wherein the oligosaccharide has up to 36 sugar moieties.

17. The process of claim 16 wherein the oligosaccharide is selected from the group consisting of maltose, maltotriase, maltotetrose, maltopentose, maltohexose, maltoheptose, and raffinose.

18. A stabilized hemoglobin product according to claim 1, complexed with carbon monoxide, said complex in aqueous solution being capable of withstanding temperatures of up to 60°C for ten hours without substantial denaturation or decomposition.

19. A stabilized CO-hemoglobin complex according to claim 18 wherein the hemoglobin units are stabilized in the T-conforma-tion.

20. A stabilized CO-hemoglobin complex according to claim 19 in lyophilized form.

21. A stabilized CO-hemoglobin complex according to claim 19 wherein the CO-hemoglobin complex molar ratio is about 4:1.

22. A process of preparing a hemoglobin product suitable for use as a blood substitute and free from transmissible viral infection, which comprises pasteurizing the carbon monoxide stabilized hemoglobin complex of claim 19 by warming a solution thereof to appropriate pasteurization temperatures for a time sufficient to cause effective deactivation of any viral contaminants of said complex and subsequently reacting the complex with oxygen to convert it to an oxy- or deoxy- hemoglobin complex or a mixture thereof.

23. Pasteurized, viral inactivated blood substitute containing conformationally and dissociatively stabilized oxy-or deoxy- hemoglobin or a mixture thereof, prepared by the process of claim 22.

24. A process for preparing met- hemoglobin complex suitable for use as a blood substitute for cyanide scavenging, which comprises oxidizing the pasteurized oxyhemoglobin of claim 23 under appropriate conditions.