new artificial oxygen carriers made of pegulated polymerised pyridoxylated porcine haemoglobin...

Comparative Biochemistry and Physiology Part A 132(2002) 185–191

1095-6433/02/$ - see front matter� 2002 Elsevier Science Inc. All rights reserved.PII: S1095-6433Ž01.00546-3

New artificial oxygen carriers made of pegulated polymerisedpyridoxylated porcine haemoglobin(P Hb)�4

Wolfgang K.R. Barnikol *, Oswald Burkhard , Harald Poetzschke , Ulrike Domack ,a,b, b a,b b

Stephanie Dinkelmann , Stefan Guth , Bernd Fiedler , Birgit Manzb b b b

Bereich Klinische Physiologie, Universitaet WittenyHerdecke, Alfred-Herrhausen-Strasse 50, D-58455 Witten (Ruhr), Germanya

SanguiBioTech AG, Alfred-Horrhausen-Strasse 44, D-58455 Witten (Ruhr), Germanyb ¨

Received 10 January 2001; received in revised form 11 April 2001; accepted 17 April 2001

Abstract

Oxygen-carrying plasma expanders are designed for use as iso-oncotic ‘blood substitutes’ to combat oxygen deficienciescaused by blood loss. In contrast, a hypo-oncotic artificial oxygen carrier can be added to existing blood — as a ‘bloodadditive’. It has potential therapeutic use for deficiencies of oxygen which are not entailed by blood(volume) lack, andcan therefore not be treated by a ‘blood substitute’, e.g. anaemias, local ischaemias and their complications such asstroke or myocardial infarction, or lack of oxygen in tumours, reducing the effectiveness of anti-cancer treatments byirradiation or chemotherapy. For such a novel approach haemoglobin-based oxygen-carrying additive, the haemoglobinmust be highly polymerised in order to decrease the oncotic pressure, which can be received many times lower comparedwith smaller molecular size haemoglobins. Our aim is to produce haemoglobin polymers with narrow distributions ofmolecular weights of approximately 1 000 000 gymol, preferably produced in high yield and at low cost. But polymerisinghaemoglobin by cross-linking normally results in a so-called percolation distribution of molecular weights, with a largeamount of insoluble material, and with only poor yields of the desired polymers. A newly developed one-vessel synthesisprocedure, which includes a controlled marked dilution of the synthesis medium during the cross-linking reaction, enablesyields of polymerised haemoglobin(P Hb) of over 80 %. Those preparations are easy and cheap to perform at large4

scales. P Hb hyperpolymers(the high molecular moiety of P Hb) are suitable for an oxygen-carrying blood additive:4 4

their oxygen-binding properties are sufficient, they are fully compatible with human blood plasma, and at the intendedtherapeutic concentration of approximately 30 gyl oncotic pressures are very low, and the impact on blood viscosity istolerable.� 2002 Elsevier Science Inc. All rights reserved.

Keywords: Artificial oxygen carrier; Blood additive; Haemoglobin hyperpolymers; Synthesis; Oxygen binding properties; Oncoticpressure; Viscosity; Bio-compatibility

1. Introduction and problems

Stimulated by great need, considerable efforts

� This paper was presented as part of ISOTT2000 held inNijmegen, The Netherlands, August 20–25, 2000. The Organ-izer was Dr Berend Oeseburg.*Corresponding author. Tel.:q49-2302-915203; fax:q49-

2302-915201.E-mail address: [email protected]

(W.K. Barnikol).

are made world-wide to develop(iso-oncotic)oxygen-carrying plasma expanders(so-called‘blood substitutes’) for clinical use. There are twomajor concepts in development: Complete artificialoxygen carriers based on(oil-in-water) emulsionsof perfluorocarbons(e.g. from Alliance Pharma-ceuticals), and chemically modified haemoglobins,designed to act freely dissolved in the bloodplasma. Until now, no oxygen-carrying therapeuticdrug has obtained a market approval. Baxter

186 W.K. Barnikol et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 185–191

Healthcare in 1997 stopped any further develop-ment of its artificial oxygen-carrying plasmaexpander, which was in phase III of clinical trials.This product consisted of(monomeric) humanhaemoglobin, intramolecularly cross-linked inorder to prevent dissociation of the molecules intofunctional subunits. Meanwhile, other candidates(glutaraldehyde cross-linked human haemoglobinpolymers from Northfield Laboratories, oxidisedraffinose cross-linked(polymerised) human hae-moglobin from Hemosol, glutaraldehyde cross-linked bovine haemoglobin from Biopure, and poly(ethylene glycol) conjugated bovine haemoglobinfrom Enzon) are also in clinical phases of devel-opment (overviews in: Winslow et al., 1997;Chang, 1997, 1998; Rudolph et al., 1998; Tsuchi-da, 1998). All these haemoglobin derivatives arefurther chemically modified by polymerisation orcoupling to macromolecules to overcome the dis-advantages of monomeric haemoglobin, e.g.extravasation and vasoactive effects. However,oxygen-carrying plasma expanders can be usedonly to combat oxygen deficiencies caused bylosses of blood, they are not applicable to solelyincrease the oxygen content of existing blood.In contrast, a hypo-oncotic artificial oxygen

carrier can be applied to combat both kinds ofoxygen deficiency(Barnikol et al., 1996): primar-ily it can — as a ‘blood additive’ — be added toexisting blood, and is therefore a potential thera-peutic to combat lack of oxygen not entailed byblood volume deficiencies, e.g. anaemias, localischaemias and their complications such as strokeor myocardial infarction, or for use in tumouroxygenation as an adjuvant for anti-cancer treat-ment. Furthermore, it can also be combined witha ‘traditional’ (non-oxygen-carrying) plasmaexpander to substitute a blood loss, regarding thelack of blood volume, as well as regarding thelack of oxygen content.We have chosen porcine haemoglobin as the

basic native material for the development of ablood additive, because of its almost inexhaustibleavailability, and because of its great similarity instructure and function to human haemoglobin. Toobtain an oxygen-carrying additive, besides severalother requirements, haemoglobin molecules mustbe highly polymerised, in order to achieve asufficient low oncotic pressure at the desired ther-apeutic concentration. To obtain a low viscosity,the molecular weights must have a narrow distri-bution with an average value of approximately

1 000 000 gymol. In addition, to meet financialneeds, the synthesis should be easily performed,using cheap materials and devices, and should leadto a high yield of the desired product.Polymerising haemoglobin with(bifunctional)

cross-linkers normally results in haemoglobin pol-ymers exhibiting a so-called percolation distribu-tion of molecular weights. These polymers covera huge range of molecular weights, thus yields ofdesired molecular species are only small.

2. Materials and methods

2.1. Materials

‘BiKu’ is an aqueous electrolyte, composed of125 mM NaCl, 4.5 mM KCl, and 20 mMNaHCO (and 3 mM NaN), and ‘Ringer’ an3 3

aqueous electrolyte composed of 143 mM NaCl,5 mM KCl, and 0.45 mM CaCl (and 3 mM2

NaN ).3

Porcine haemoglobin(Hb) of high purity wasprepared from fresh blood received from a localslaughterhouse.‘PLP’ is pyridoxal-59-phosphate(3-hydroxy-2-

methyl-5-w(phosphomonoxy)methylx-4-pyridine-carboxaldehyde), ‘GDA’ is glutaraldehyde(1,5-pentandial), and ‘PEG’ is a(monofunctionalN-hydroxysuccinimidyl ester activated), polyeth-ylene glycol of 2000 gymol molecular weight.‘Pyridoxylation’ describes the covalent coupling

of PLP to haemoglobin, and ‘pegulation’ the cova-lent coupling of the active PEG to the haemoglobinpolymers.

2.2. Synthesis and preparation of haemoglobinpolymers

In brief, pegulated polymerised pyridoxylatedporcine haemoglobin was synthesised by covalentcross-linking of purified and initially highly con-centrated (320–340 gyl) pyridoxylated porcinehaemoglobin by means of the bifunctional cross-linker glutaraldehyde, succeeded by pegulation,and separation of the polymers of desired molec-ular size from the remainder of the broadly distrib-uted haemoglobin polymerisation reaction product.Initially, PLP (in an iso-molar ratio to haemo-

globin) was coupled covalently to the deoxygen-ated haemoglobin, followed by polymerisationusing GDA (in an 8.5-molar surplus) as cross-linker, and by subsequent covalent coupling of the

187W.K. Barnikol et al. / Comparative Biochemistry and Physiology Part A 132 (2002) 185–191

active PEG(6 mol per mol of Hb) to the poly-merised pyridoxylated haemoglobin. Aldehyde-derived cross-links(aldimines) were stabilised byreduction, using sodium borohydride. P Hb hyper-4

polymers were separated using preparative sizeexclusion chromatography(using ‘Sephacryl S-400 HR’ gel from Pharmacia, Freiburg, Germany,and ‘BiKu’ as the elution medium), also resultingin a removal of all small molecular size chemicals,and complete exchange of the solvent.

2.3. Analyses

Haemoglobin(native and polymerised) concen-trations were measured photometrically, using thehaemoglobin cyanide(modified Drabkin’s) meth-od. This method has been validated for the hae-moglobin polymers considered here.Molecular weights of haemoglobin polymers

(and oligomers), and their distributions were deter-mined by means of an analytical size exclusionchromatography(SEC), performed as a ‘conven-tional’ column chromatography, with photometri-cal detection of the eluate’s absorbance at 425 nm.Columns were 84 cm long and had an innerdiameter of 1 cm, chromatographic gel was‘Sephacryl S-400 high resolution’(Pharmacia,Freiburg, Germany), the elution medium was‘BiKu’ with a flow rate of 5.2 mlyh, and the platecounts were approximately 3500ym. Valid molec-ular weight calibrations were performed usingnative proteins as calibration standards, and thecorrection procedure as described earlier(Poetzschke et al., 1997).The oxygen binding characteristics of haemo-

globin hyperpolymers were measured using a spec-trophotometric thin-layer micro method anddevice, which is our own development(Barnikolet al., 1978), whereby, at a temperature of 378C,haemoglobin solution specimens were equilibratedwith gas mixtures of known composition, espe-cially carbon dioxide with 40 torr partial tension,which mimics physiological conditions. Two majoroxygen binding characteristics(overview in:Antonini et al., 1981) of haemoglobin polymersare the oxygen pressure(p) at half saturation(S)of oxygen binding sites(p ) as a measure for50

their oxygen affinity, and a mean Hill’s index(n ), derived as the steepness of the Hill function50

(log wSy(1yS)x vs. logp) in the oxygen saturationinterval from 0.4 to 0.6, describing the intramolec-ular co-operativity of oxygen binding sites.

Colloid osmotic(soncotic) pressures of aque-ous solutions of P Hb hyperpolymers were4

assessed by means of a ‘Membrane OsmometerMO (Knauer, Berlin, Germany), using ‘BiKu’ asthe solvent, and cellulose acetate separation mem-branes with nominal molecular weight limits of 20kDa (details in: Poetzschke et al., 1997).Kinematic viscosities were measured at 378C

with calibrated Ubbelohde micro glass capillaryviscometers (Schott, Mainz, Germany), P Hb4

hyperpolymers were dissolved in ‘BiKu’, or inhuman plasma. A stock solution of P Hb hyper-4

polymers in human plasma was prepared by mix-ing 3 ml of P Hb hyperpolymers dissolved in4

‘Ringer’ electrolyte, and 12 ml of fresh human(H.P.) plasma, followed by a volume reductiondown to the initial plasma volume(12 ml), usinga stirred cell ultrafiltration system(‘Omegacell’,with a nominal molecular weight limit membraneof 30 kDa, Pall Filtron, Karlstein, Germany).Bio-compatibility of haemoglobin polymers

with human plasma was tested by incubation ofmixtures of both components at different levels ofthe pH(as described in: Domack, 1997). In brief,mixtures of equal volumes of human plasma andaqueous P Hb solutions in ‘Ringer’(resulting in4

haemoglobin concentrations between 20 and30 gyl) were titrated with lactic acid to pH valuesbetween 6.8 and 7.6, and after at least 30 min atroom temperature and at 378C, respectively,incompatibilities can be assessed as decreases ofhaemoglobin contents, caused by precipitated com-plexes of haemoglobin and plasma proteins.

3. Results

The whole synthesis of pegulated polymerisedpyridoxylated porcine haemoglobin can be carriedout in a one-vessel procedure. The formation ofan unfavourable percolation distribution of poly-merisation degrees can be avoided by controlleddilution of the reaction mixture during the cross-linking reaction of the haemoglobin molecules. Asan example, Fig. 1a shows the molecular weightdistribution of a typical raw product, producedwith the mentioned synthesis procedure in anoverall yield of 74%. The molecular weight distri-bution of this synthesis product is characterised bya defined upper limit of approximately 5 000 000gymol, this limit is adjustable by varying thereaction conditions. The area under the curve(AUC) fraction of polymers having a molecular


Table 1Oxygen partial pressures at half saturation of haemoglobinoxygen binding sites(p ) and a mean Hill’s index(n ) as50 50

the fundamental oxygen binding properties of human and por-cine native haemoglobin, and of porcine haemoglobin derivedP Hb, all assessed at physiological conditions of pH(7.4 in4

solution, and 7.2 inside erythrocytes), of carbon dioxide partialpressure(40 Torr), and temperature(37 8C)

Haemoglobinyderivative p (Torr)50 n50

Human Hb-within full blood 25 2.5(Stripped) human Hb 15 2.5(Stripped) porcine Hb 15 2.5P Hb hyperpolymers4 21 2.0

‘Stripped’ haemoglobin is purified from organic phosphates,especially 2,3-bisphosphoglycerat(DPG), and adenosine tri-phosphate(ATP).

Fig. 1. Original size exclusion chromatograms of(a) a typical P Hb synthesis product, and(b) the fraction of hyperpolymers(polymers4

with molecular weights greater than 700 000 gymol). Attached is a molecular weight(M) scale, so these chromatograms show theartificial haemoglobin polymers MWD.E is the optical density,V the elution volume, B vitamin B (used as an internalV indicatore 12 12 t

standard substance), and the percentages shown are the values of the relative AUC, representing the respective fractions of the absoluteyield.

weight of greater than 700 000 gymol of thismodel raw synthesis product is approximately39%, where 700 000 gymol is an arbitrarily chosenlimit between high molecular weight polymers(hyperpolymers), which are suitable as an oxygen-carrying additive, and the remaining low molecularweight polymers(oligomers) and uncross-linkedhaemoglobin monomers. Fig. 1b shows the analyt-ically determined molecular weight distribution ofthe hyperpolymers fraction from this batch, afterthey were separated from the raw product by apreparative size exclusion chromatographyprocess.A very important property of P Hb hyperpoly-4

mers as a candidate oxygen-carrying blood addi-tive is their ability to reversibly bind oxygen. Table1 shows their major oxygen binding characteristics,compared with human and porcine haemoglobinunder different conditions. The oxygen pressure athalf saturation(p ) of P Hb hyperpolymers ranges50 4

from 19 to 24 Torr, and Hill’s index(n ), as a50

measure of co-operativity between the oxygenbinding sites, ranges from 1.8 to 2.2. These values

are almost independent from the molecular size ofthe polymers.Other important physico-chemical properties of

P Hb hyperpolymers are their oncotic(colloid-4

osmotic) and viscosity properties. Fig. 2 showsthe results of oncotic measurements from twobatches of P Hb hyperpolymers. At a concentration4


Fig. 2. The oncotic pressure(p ) of aqueous solutions of twoonc

typical batches(d and s) of P Hb hyperpolymers(M)4

700 000 gymol) as a function of the haemoglobin concentra-tion (c Hb). The value of plasma concentrationsm

therapeutically desired is 30 gyl, the dashed line(p BL) indi-onc

cates the value of human blood.

Fig. 3. The kinematic viscosity(n) of solutions of typical P Hb4

hyperpolymers(d ands) dissolved in ‘BiKu’ and in humanplasma(PL) as a function of the haemoglobin concentration(c Hb). The dashed line(nBL) indicates the value of humanm

blood.

Fig. 4. So-called acidic bio-compatibility titration curves of themodel P Hb hyperpolymers batches.c Hb is the P Hb con-4 m 4

centration, pH the potentia hydrogenii,d ands are the meas-ured values, the lines indicate the calculated values.

of 30 gyl (which is considered to be approximatelya sufficient concentration for therapeutic uses), theoncotic pressure is approximately 3 mbar, whichis only approximately 9% of 35 mbar, the physi-ological value of human blood plasma. Viscositymeasurements from the same model P Hb hyper-4

polymers solved in the electrolyte BiKu, as wellas in human plasma, are depicted in Fig. 3.Regarding again a concentration of 30 gyl, thekinematic viscosity of the solutions in electrolyteis only approximately 1.4 cSt, also the hyperpo-lymers containing plasma shows a lower viscositythan whole blood, which(at comparable conditions— but whole blood is a non-Newtonian fluid!) isapproximately 3.4 cSt.A further important factor regarding the use of

artificial haemoglobin hyperpolymers as intravas-cular oxygen carriers is their bio-compatibilitywith the recipient’s plasma proteins. Fig. 4 depictsthe results of so-called compatibility titrations, thediagram shows the haemoglobin contents in iso-volaemic mixtures of the two batches of P Hb4

hyperpolymers mentioned above, and fresh humanplasma at several pH values after incubation.Within the range of error, all measured values ofhaemoglobin concentration are equal to the theo-retical value, calculated by using the initial hae-moglobin solution content, as well as the degreeof dilution by blood plasma and lactic acid solu-

tion. As a result, P Hb hyperpolymers are fully4

compatible with human blood plasma.

4. Discussion

The described one-vessel synthesis procedurefits the cost-saving financial requirements of thehealth services. It enables a very easy and cheapsynthesis of the artificial oxygen carrier, especiallyaiding the necessity for an ab initio sterile prepa-


ration, which is necessary because a final sterilis-ation of the sensitive product is not possible. Alsothe overall yield of more than 75% of suitable rawmaterial, as achieved by the special dilution proc-ess during cross-linking, helps to economise onthe manufacturing process. Furthermore, the wholeraw material is suitable for use as oxygen carriers:The high molecular weight haemoglobin hyper-polymers may be used as an oxygen-carrying bloodadditive. These polymers, because of their definiteupper molecular weight limit, can easily be sepa-rated from the raw product by preparative sizeexclusion chromatography or ultrafiltration. Theremainder, the low molecular weight moiety, maybe used as an oxygen-carrying blood volumeplasma expander. But, the oxygen-carrying bloodadditive may also be used to substitute lost bloodby simultaneous application with a classical(non-oxygen-carrying) plasma expander, for instance,with a serum albumin solution.By changing the synthesis reaction conditions it

is possible to vary, and adjust the oxygen bindingcharacteristics of the product, i.e.p and n50 50

values. Hill’s index should be kept as high aspossible, because this is advantageous for both,the uptake of oxygen in the lung, and the releaseof oxygen in the tissue. Regarding the oxygenaffinity of artificial oxygen carriers, as comparedwith the p value of the recipient’s blood, one50

can differentiate artificial oxygen carriers to beisoxybar, hypoxybar, or hyperoxybar. Consideringman, for instance, an isoxybar artificial oxygencarrier has ap of approximately 25 Torr, this50

p value of a hypoxybar carrier is lower, and of50

a hyperoxybar carrier higher than 25 Torr. Bycomparing the effects of different artificial oxygencarriers on intravascular oxygen partial pressureswithin various tissues, Conover et al.(1999) havedemonstrated, that hypoxybar carriers are mosteffective in delivering oxygen to tissue. Therefore,we prefer this kind of artificial oxygen carrier.Polymerised haemoglobins, especially hyperpo-

lymers, are not easy to maintain in solution,especially interactions with the proteins of bloodplasma can lead to precipitation, if both compo-nents are not compatible, a point that until nowwas rarely considered by others. We have devel-oped a test, to prove this special bio-compatibilityof haemoglobin polymers(Domack and Barnikol,1996; Domack, 1997). As demonstrated in Fig. 4,the hyperpolymers prepared are fully compatiblewith fresh human blood plasma, which is achieved

by pegulating the polymers. However, pegulationof haemoglobin leads to distinct increases in oncot-ic pressure and viscosity(Vandegriff et al., 1997).As shown in Figs. 2 and 3, the degree of pegulationresulting from our synthesis procedure is suitablefor both, oncotic pressure and viscosity, to be lowenough at the desired therapeutic concentration ofapproximately 30 gyl.

Acknowledgments

We express our thanks to Karola Krausse, BTA,Silke Mischliwietz, Biol.-Lab., Birgit Wintzek,BTA, Bianca Schnettler, BTA, and Tanja Claus-meier, BTA, for carefully planning, performing,and evaluating the experimental work.

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new artificial oxygen carriers made of pegulated polymerised pyridoxylated porcine haemoglobin...

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