Effects of chemical modifiers on recombinant factor VIII activity

Thrombosis

Pergamon

Research, Vol. SO, No. 3, pp. 247-254, 1995 Coovrieht 8 1995 Elsevier Science Ltd F&&d-in the USA. All rights reserved 0049.384W95 $9.50 + .oO

0049-3848(95)00173-5

EFFECTS OF CHEMICAL MODIFIERS ON RECOMBINANT FACTOR VIII ACTIVITY Fiona MANNING, Ciaran 6 FAGAIN,Richard O’KENNEDY & Barry WOODHAMS ’ Applied Biochemistry Group, Schoolof Biological Sciences.Dublin City University, Dublin 9, Ireland & ‘Dade Produktions AG,Bonnstrasse9, 3186 Dudingen, Switzerland. (Received 2 June 1995 by Editor El. Gaffney; revised/accepted 16 August 1995)

Abstract Factor VIII performs a critical role in the blood coagulation cascade but is extremely labile. We have carried out chemical studies on a fully functional recombinant Factor VIII (rFVII1)using a variety of protein modifying agents,some of which were bifunctional. Thiol-specific maleimides cause no activity loss despite a substantial decrease in #VIII’s free thiol content. Mild oxidation procedures had either neutral or adverse effects on activity. Amino-specific reagents led to significant lossesof rFVII1procoagulant activity. It appears that free amino groups are essentialfor Factor VIII’s function while some at least of its many free thiol groups are not. Noneof these derivatives was any less labile than unmodified rFVIII. Systematicchemical modification of important proteins in this way can give useful insights into appropriate protein engineering strategies.

Factor VIII (FVIII) is a large glyco rotein which is deficient or functionally abnormal in the bleeding disorder haemophilia A P1). It circulates in normal plasma as part of a complex in which it is non-covalently bound to von Willebrand factor (vWF). To articipate in coagulation, FVIII is released from vWFas a heterotrimer by thrombin (2P Inactivation follows dissociation of one subunit from the trimer (3) or further proteolysis by thrombin or activated Protein C (4). FVIII is stabilised in viuo by vWFand becomeshighly unstable in its absence. Chemical derivatization of therapeutic proteins can lead to desirable alterations in function and can be a useful complement to genetic manipulation (5). Here we describe a systematicchemical study on a recombinant FVIII(rFVII1).Weexaminethe effects of specific Key words: Factor VIII, recombinant protein, chemical modification Correspondingauthor: Dr Ciarbn 6 FAGAIN, Applied BiochemistryGroup, Schoolof Biological Sciences,Dublin City University, Dublin 9, Ireland. 247

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chemical modifiers on rFVIII’s activity and we investigate whether modification of certain groups can alter the stability of this crucial protein. EXPERIMENTAL PROCEDURES MATERIALS

& GENERAL

METHODS

RecombinantFactor VIII (rFVII1)was a gift from Baxter Hyland (California, USA).[The 980-residue B-domain is removed by thrombin during Factor VIII activation and is not associatedwith Factor VIII’s procoagulant function (6).] Factor VIII Chromogenic Assaykit (281068), Coag Cal N (291040) and Factor VIII Deficient Plasma (281042) were gifts from Dade Produktions AG (Dtidingen, Switzerland). Chemicalsfor modifications were obtained from Sigmaexcept for Bis-(maleimido)-methyl ether (BMME)(Boehringer Mannheim),hydrogenperoxide and acetone (BDH).SephadexG-25 was purchased from Pharmacia. Buffers used were 10mMTris/HCl pH 7.5, containing 0.9% NaCl,3mM CaC1,and 0.05%(w/ v) sod’mm azide and 1OmMbarbital buffer pH 7.5, containing 0.9%(w/v) NaCl,3mMCaCl,and 0.05%(w/v) sodium azide. Factor VIII procoagulant activity wasmeasuredby a kinetic chromogenic assay(Dade Produktions AG; absorbancesread at 405nm at 15 second intervals up to 2 minutes) miniaturised for use in a 96-well microtitre late (volumes reduced to 25% of recommended).Sampleswere diluted 1:31in 0.9%(w/pv) NaC!before assay.Immunoadsorbed Factor VIII-Deficient Plasma and CoagCal N were used as negative and positive controls, respectively, in each set of Factor VIII activity determinations. For thermoinactivation studies,solutionsof native and modified rFVlII were incubated in a 55°Cwaterbath. Sampleswere withdrawn at 0, 2, 5, 10,20, 40 and 60min stored on ice and assayedfor FVIII activity in 0.9%(w/v) NaCl.Positive (CoagCal N) and negative (FVIIIDeficient Plasma) controls, as well as rFVlI1 diluted in the same buffer as the modifying reagent, were incubated in each experiment. For amino-specific reagents, rFVII1was transferred at 4°Cto barbital buffer. This contains no free amino groups which might interfere with the reaction, rFVII1was applied in lOOl.~laliquots to a Pasteur pipette packedwith SephadexG-25 and eluted usin barbital buffer. The protein concentration of each fraction was estimated by BCAassay47) and a working dilution was calculated. The free thiol content of rFVII1wasestimated before and after BMMEmodification (see below) using Ellman’s reagent [5,5’-dithio-biss(22nitrobenzoic acid); DTNB;(a)] 501.~1 of rFVII1or cysteinestandard was addedto 50~1of 3mMDTNBin O.lMphosphate buffer pH 7.2 and incubated for 15min at room temperature. Absorbancewasthen read at 414nm and the cysteine content calculated usin the molar absorption coefficient of the DTNB-cysteine complex [Q~= 14 150M-‘cm-‘; MODIFICATIONS

WITH

CROSSLINKERS

rFVII1in Tris was reacted with two thiol-specific bis-maleimide crosslinkers (9) dissolved either in acetone (for BMME), or dimethylformamide (for N-N-bis[Smaleimidopropionyll-2-hydroxy 1,3-propanediamine (BMPPD)). Reactiontook place at room temperature, for 10 and 60 min and was stopped by addition of 5~1 100mM 2mercaptoethanol. FVIII activity was measured. rFVII1in barbital wasreacted with final concentrationsof O-40mMof the bis-imidate in dimethyl formamide (DMF)at 4°C(10). Reatt’ion proceededfor 30 min before termination by addition of l/lo volume of ice cold Tris buffer. FVIII activity was measured. rFVII1 in barbital was reacted with O-3mM glutaraldehyde at 4°C for lh, being

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terminated as above. A 25mM stock solution of the heterobifunctional, N-(y-maleimidobutyryloxy) succinimide [GMBS,(II)] was prepared in DMF.From this, final concentrations of 100pMand 250pMwere reacted with rFVII1in barbital at either 4°Cfor 10min or room temperature for 30min. Reaction was terminated by addition of I/IO volume of ice cold Tris buffer with a further 5 min incubation at 4°C.FVIII activity was assayed. rFVII1in barbital was reacted with a 100-fold molar excessof the heterobifunctional succinimidyl 4-(N-maleimidomethyl) cyclohexane carboxylate, [SMCC; (12)] in dimethylformamide for 1 hour at room temperature (with and without stirring). I/IO volume of ice-cold Tris buffer was added and sample was stirred for an additional 30min to terminate the amino-specific reaction. FVIII activities were determined. USE OF PROTEIN

MODZFYING

REAGENlS

rFVIII in barbital was reacted with cyanuric chloride-activated olyethylene glycol (13) to final molar excessof 0.25 and 2.5 over rFVII1lysines[approx 75: P14)]. Reaction took place at room temperature for lh before being terminated by addition of l/10 volume of icecold Tris buffer. FVIII activity assaytook place after a further 15min incubation. 1mMstock solutions of C&O, and o-phenanthroline (o-ph) (15) were prepared in Tris. From these,various combinations of each (lOpM,~OJ.LM and 50pMCuSO,and 2$M, 5OpM and 100pMo-ph) were reacted with rFVII1at 4°Cfor 20min. This wasto promote thiol group oxidation andpossibleformation of additional disulphide bridges betweensuitably-positioned cysteines FVIII activity was then assayedand thermoinactivation studies performed. Final concentrations in the range O-1M H,O,were reacted with rFVII1on ice for 15 min, lh and 2 h in an attempt to oxidise methionine residues to sulphoxides (16). Reaction was terminated by addition of 2% (w/ v) cat a1ase enzyme solution (lmg/ml) to oxidise remaining peroxide. After 15 minutes’ further incubation on ice FVIIIactivity was measured. rFVII1in barbital was reacted with final concentrations of 0, 0.25mM,lmM, and 6mM 22Iminothiolane (2-IT) in water at 4°Cfor 2h. 2-IT adds to free amino groups introducing a thiol (17). 10%volume of ice cold Tris buffer wasaddedto terminate reaction. FYI11activity was assayedand thermoinactivation was carried out. Also,rFVII1was treated with 2-IT and then reacted with either 40mMH,O,or with o-ph (100pMo-ph/50pM CuSO,)(15). This was to promote disulphide bond formation betweenthiol groups introduced via 2&IT, and free thiol groups on rFVII1.FVIII was assayedand its thermoinactivation studied, RESULTS Wehave found that loss of Factor VIII procoagulant activity at 55°Coccurs via a first order exponential process,consistent with a unimolecular loss of activity (k= 0.23+ 0.02,average of 6 determinations). The concentrations of the cross-linkers used were in multiples of the moles of lysine (75/mol FVIII) or cysteine(19/mol FVIII)residuespresent in the recombinant FVIII molecule (6,14). Thus, the concentrations were in the PM to mM range. Note that all chemical reactions were terminated by addition of an excessof that reactive group (e.g. mercaptoethanol’s -SH groups removed any remaining maleimide moleculeswhich had not reacted with FVIII) and that each sample was diluted 1:31in 0.9%NaClprior to assay,Thus, there should be no modifier carry-over effect on the procoagulant assay.Note also that the 100%FVIlI control in each experiment in Table 1 refers to FVIII treated exactly as for its modified counterpart except that the reagent was omitted,

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TABLE1. ACTIVITIES OFrFVII1SAMPLES FOLLOWING CHEMICAL MODIFICATION

*A405 values in the chromogenic assayare a direct measureof FVIIIprocoagulant activity. Eachassaywasperformed in triplicate and each experiment in duplicate or triplicate. Positive and negative controls included in each experiment performed within manufacturer’s specifications. THIOL-SPECIFIC

REAGEiWS

N-substituted bis-maleimides are specific and mild bifunctional crosslinkers for sulphydryl groups, Two of these were used in an attempt to form crosslinkswithin rFVII1. Neither BMMEnor BMPPDin the range O-100pMhad any adverse effect on rFVII1activity (Table 1). Neither compound,however, increased rFVII1thermostability. The number of free thiols in rFVII1was estimated before and after BMMEmodification using Ellman’s reagent. BMMEtreatment decreasedthe free thiol content from 20yMto 8pM.This implies that BMME has reacted specificallywith the thiol groups of cysteineresidues.Thesemodifications do not affect rFVIII’s procoagulant activity.

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REAGENTS

Three of the bis-imidate series - dimethyl adipimidate, dimethyl pimelimidate and dimethyl suberimidate - were reacted with rFVIII in barbital buffer. Each resulted in almost complete lossof FVlII activity (Table 1). Denaturation studies were carried out to determine whether the remaining procoagulant activity showedincreasedthermostability. No increase was detected. Glutaraldehyde concentrations >lmM resulted in loss of rF’VlI1activity. Eachof the two heterobifunctional reagentscontained an amino-specific succinimide moiety at one end and a thiol-specific maleimide at the other. Addition of either causedan immediate loss of FVIIIactivity, similar to that observedwith other amino-specific reagents. Cyanuric chloride-activated polyethylene glycol (PEG)was also employed to modify free amino groups of #VIII. Proteins to which PEGhas been attached have been shownto be stable and active in organic solvents (18) and to be less antigenic and allergenic (5). However, only a small level of activity remained after reaction and thermoinactivation studies showedno increase in stability. 2-Iminothiolane (22IT) reacts with lysine amino groups, introducing an additional thiol group (11). Disulphide bonds may be formed when the derivative is subjected to oxidation if a second free thiol lies closelyenough.Addition of Z-IT in the range O-6mM to rFVII1did not causeany loss of activity (Table 1). Thermoinactivation studies showedthat the Z-IT-modified rFVIl1was no more stable than untreated rFVII1.The second stage of this experiment involved oxidation of the thiolated derivative in an attempt to form additional disulphide bridges. Two reagents were used, hydrogen peroxide and the ophenthroline/copper chelate (o-ph/Cu). Peroxide destroyed rFVII1activity but addition of o-ph/Cu did not result in loss of activity. Whileneither modification step causedany major loss of activity, the stability of rFVII1was not enhanced. DISCUSSION Aibumin is often included as a stabilizing agent in FVIII solutions but was not used here. It was omitted from the FVIII solutions used for chemical modification since it would have interfered with the reaction. Albumin was also omitted from post-modification FVIIIbuffers and dilutions. The e-amino group of lysine residuesis strongly nucleophilic and is ideal for selective modification. Amongthe functional groups of amino acids,the thiol group of cysteineis the strongest nucleophile, reacting with most thiol-specific agents at an alkaline pH (19). However, because rFVII1 contains a number of activation sites where arginine residues predominate, one must avoid reagents which would alter these sites in any way. Carboxylspecific reagents were not used sinceclusters of ne atively charged carboxyl groups bind the calcium ions which are essential for FVIII activity f 1). FVIII’spH tolerance is narrow. Inactivation occurs at pHvalues less than about 6 and greater than 8 (20,21). This reduced the range of modifiers which could be used, as many reagents act optimally outside this narrow pH range. This was particularly important for the heterobifunctional cross-linkers, where each end of the molecule is selectively activated at different pH values. The range of such compounds which could be used was therefore severely restricted. Most chemical modifications of proteins are performed at protein concentrations about ten-fold higher than used here. Whilethe clotting activity of rFVII1 used was high, its protein concentration was 0.150.3mg/ml (approx. 0.9-1.8pM). The amount of rFVII1available was greatly restricted, so concentration of the protein could not be carried out. This, together with activated FVIII (FVIIIa)‘s extremely narrow pH tolerance,

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forced us to operate within very limited conditions, some of which were not ideal for the reagents in question, Imidoesters, for example, react optimally with unprotonated amino groups at mildly alkaline pH values which FVIII will not tolerate. The low protein concentration also prevented us from characterizing the chemical derivatives produced: we could not ascertain the degreeof change in the number of free amino groups, for example. Likewise,biophysical approachesto FVIII unfolding etc. could not be undertaken, Our only experimental parameter, therefore, was FVIII procoagulant activity as determined by chromogenic assay. However, this is the activity that will be primarily important in any diagnostic or therapeutic application of rFVII1or any derivative, Due to scarcity of time and materials, and to operational limits in thermoinactivation experiments, procoagulant activity was determined at a single FVIII dilution which had been previously demonstrated to give satisfactory results in the kinetic chromogenic assay.Activity of each sample was calculated relative to a control FVIIItreated in exactly the sameway except that the modifier was omitted. Assay of a number of FYI11dilutions following chemical modification would have permitted determination of parallel dose-responserelationships for native and modified FVIIIs and may have been more informative. There is a notable difference betweenthe effects of the amino- and the thiol-specific reagents on rFVII1.With the exception of 2-IT, all of the amino-group modifers, whether cross-linkers or not, inactivate most of the procoagulant activity. It is not clear why 2-IT alone did not inactivate rFVII1.In contrast, the thiol-specific reagents did not lead to rFVII1 inactivation. This suggeststhat somefree amino groups are essentialfor rFVlI1integrity and activity. At least some of the numerous free thiol groups can be modified, however, without adverse effects. Free thiol (cysteine) content decreasedfrom 20pM to 8f.1,M following BMME treatment. This represents a reduction of 60%in the content of titratable cysteineswith only a 14%reduction in procoagulant activity (Table 1). If one assumesthat rFVII1(no B-domain) has a molecular mass of 17OkDa(l), our working solution of rFVII1 (0.3mg.ml11)was approximately 1.8pM.rFVII1has a cysteine count of 19 (6) or 34pM. This is considerably greater than the figure of 20yM determined above. DTNBmay not accessall cysteinesof a particular protein: it reacted with all 4 cysteinesof denatured ovalbumin but failed to detect any in the native form (22). Regardless of the discrepancy between calculated and experimentally-determined cysteinecontent of rFVII1,our results demonstrate that most of rFVIII’s free thiol groups can be modified without significant adverse effects on procoagulant activity. These findings contrast with those of Austen (23). He noted that thiol-specific treatment of bovine FVIIIled to its inactivation. Austen’streatments included iodoacetamide and para-chloromercuribenzoic acid (pCMB).The inactivation of pCMB-treated FVIII could be reversed by addition of cysteine(a reducing agent). Thereagents used by Austenare more severe than the mild maleimido esters.However,Austen’sstudy most likely was carried out on the FVIII/vWFcomplex rather than on pure FVIII. Contamination of FVIII with vWFwas a significant problem up to the 1980s(24). Hence A&en’s results, dating from 1970,should perhaps be interpreted with caution. Oxidationreactions were either neutral (attempts to create newdisulphide links with o-ph/Cu) or deleterious (attempts to oxidise methionineswith H,O,).There are a number of inactivation sites present in the FVIIImolecule. Methionineis present in the inactivation site Arg336-Met337 of native FVIlI (14). We attempted to oxidise this at 0°C to the bulky sulphoxide form and, thereby, prevent inactivation cleavage.H,O,is quite a strong oxidant, however, and may have reacted with residues other than methionine. Austen (23) also used H,O, on beef ‘FVIII’ (see above) at 20°C and found that concentrations >O.OlMcaused irreversible inactivation.

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Factor VIII undergoes many cleavages,some of which are necessaryfor molecular activation while others lead to inactivation. The activation cleavagesites at least must be preserved in any functional derivative which is to be prepared. Since it is also a large and complex molecule, any successfulcross-linking or other modification may be extremely subtle and represents a considerable challenge. Nonetheless,our experiments have shown a qualitative difference in functional importance between different amino acid side chains within FVIII. Amino groups seemto be essential for FVIII activity while thiol groups may be modified without adverse effect. These findings may prove significant for a further characterisation or understanding of this critical procoagulant component, or for its protein engineering. Acknowledgment

Wethank Baxter Diagnostics(now DadeProduktions) AGand the Irish American Partnership for financial support. REFERENCES 1.MANNING, F.,6 FAGAIN, C.& O’KENNEDY, R.FactorVIII:structure,functionandanalysis.Biotechnol. Adv.11 199114, 1993 2. LOLLAR, P. & PARKER, C.G. Subunit structure of thrombin-activated Biochemistry 28 666-674, 1989

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