CA1251152A - Ultrapurification of factor viii - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A method of preparing high purity procoagulant protein comprising the steps of (a) adsorbing a VIII:C/VIII:RP complex from a plasma or commercial concentrate source of factor VIII onto agarose beads bound to a monoclonal antibody specific to VIII:RP, (b) eluting VIII:C with a salt solution, (c) adsorbing the eluted VIII:C on an animohexyl agarose column and eluting the VIII:C with a salt solution.

Description

I ~ZS~5~

BACKGROUND OF THE INVENTION

Field of the Invention _ .

This invention relates generally to a method of separa-ting and purifying factor VIII procoagulant activity protein, More specifically, high purity factor VIII procoagulant activity protein is separated from von Willebrand Factor by a two step`
chromatographic adsorption and concentration technique from plasma or concentrate, Description of the Prior Art The isolation of the antihemophilic factor from blood plasma has been described in the literature. The precise struc-ture of the antihemophilic factor, also referred to as factor VIII, has not yet been identified, due in part, to the unavail-ability of sufficient quantities of pure material with which to conduct further studies. The limited availability of pure mate-rial and its existence in a dilute state has also hindered its use in therapeutic applications, Factor VIII procoagulant activity protein functions to correct the clotting defect in hemophilic plasma. It circulates in plasma complexed with the von Willebrand factor protein. The latter can alter the platelet function defect in von ~illebrand's disease, That portion of the factor VIII von l~illebrand factor complex having coagulant activity is referred to as factor VIII
procoagulant activity protein, factor VIII - clotting activity or si~ply VIII:C (the designation of "VIII:C" will be used herein-:125:1L15;2 after to identify the portion of the factor VIII molecule withsuch clotting activity.) The other portion of the factor VIII
von Willebrand factor complex having the ability -to correc-t the platelet function defect in von ~illebrand's disease is referred t as von Willebrand factor, factor VIII - related antigen, VIIIR:Ag, VIII:RP factor. (The description `'VIII:RP" will be used herein-after to identify the platelet correction function of the factor VIII molecule). Although yet unproven, there is evidence to sup-port the conclusion that VIII:C exhibits properties and the behavi of a small molecule which is combined with VIII:RP as a non-covalent complex. There is also a basis for the contention tha-t the properties associated with both VIII:C and VIII:RP may also be a single molecule which under appropriate conditions may be cleaved, yielding two fragments.
In view of the need for identifying the structures of the factor VIII/von Willebrand factor complex, VIII:C and VIII:RP
and the important pharmaceutical value of the coagulant activity ascribable to VIII:C, numerous attempts have been made to purify factor VIII and to separate ana concentrate VIII:C and VIII:RP.
The techniques used are based generally on either immunoadsorptio or ion exchange chromatography. Such techniques as heretofore used have had limited success due to the difficulty of desorbing the proteins from the charged ionic material in an undamaged con-dition or recovering same in suitable guantities.
One such method for separating VIII:C from VIII:RP
utilizing immunoadsorbent chromatography has been reported by E. G. D. Tuddenham et al, "The Properties of Factor VIII Coagulan Activity Prepared by Immunoadsorbent Chromatography", JO~RNAL OF
LABO~TORY CLINICAL ~EDICI~E, Vol. 93, p. 40 (1979). The repor~e lZ5~5~2 1 ethod i5 a one-step separation of VIII:C from nearly all VIII:RP ¦
and from most other plasma proteins employing a chromatoyraphic column packed with agarose beads to which polyclonal antisera to VIII:RP (anti-VIII:RP) are coupled. Factor VIII/von Willebrand factor con-taining plasma is passed through the column which adsorb both VIII:C and VI~I:RP. Other unwanted plasma proteins are removed from the column by washing with buffered saline solution and the desired VIII:C is obtained by subsequent elution with a calcium-ion grad;ent. Althou~h it is stated to be an improvement in both purity and yield of VIII:C, when compared to the previousl known methods, it is also stated that the resul-ting product also contains VIII:RP and other plasma proteins. Such contaminants may be attributable to the use of polyclonal antisera bound to the agarose beads. Since a majority of the immunoglobulins from which the antisera are constituted are not specific to VIII:RP, the effective number of sites where antibodies specific to VIII:RP
may be bound to agarose is relatively small due to competition between the antisera for a finite number of bonding sites on the agarose.
Another method for separating VIII:C from VIII:RP and ristocetin co-factor by a chromatographic technique employing aminohex~l-substituted agarose has been described by D. E. G.
Austen, "The Chromato~raphic Separation of Fàctor VIII on Amino-hexyl Sepharose," BRITIS~ JOURNAL OF HAEMATOLOGY, Vol. 43, p. 669 tl979). The described method is stated to be an improved method .
for the component parts of both human and porcine factor VIII~von Willebrand factor. This method, ho~ever, also suffers from the fact that contaminants are present in the resultin~ product. In both the Tuddenham et al and Austen methods a contaminated product ~2~L5~ l which is more dilute than is normally desired, is formed.
Hence, it is clear that there still exists a need for an improved method for separating and purifying VIII:C from VIII:RP using plasma or concentrates. Therefore, it is an object the present invention to satisfy such a need.

SUMMARY OF THE INVENTION

The present invention relates to a method of separation of the co~ponent molecules of the factor VIII/von Willebrand Eacto comple~, VIII:C and VIII:RP, and the puriEica-tion and concentra-tion of the pro-coagulant activity protein VIII:C. The method achieves the object of producing highly purified VIII:C using a two step procedure.
The first step involves immunoadsorption of factor VIII
from plasma or a commercial concentrate. The adsorbent employed comprises a monoclonal an-tibody specific to VIII:RP which is bound to a suitable substrate such as, agarose beads. After the VIII:C/VIII:RP is initially adsorbed, the substrate particles are washed extensively with a buffer solution to remove unadsorbed protein. The adsorbed material is then treated with a calcium ion containing solution to elute the aasorbed VIII:C. The VIII:RP
portion remains adsorbed on the anti-VIII:RP bound material. At this point about 40-60% of the VIII:C initially adsorbed is re-covered in a highly purified state. However, the procoagulant activity protein recovered, although extremely pure, i.e., largely free ~rom contaminants, is too dilute to be of si~nificant thera-peutic value.

~ 125115~ 1 The second step of the present process is directed to substantially concentrating the recovered purified VIII:C usiny a technique which may be characterized as affinity chromatography.
The VIII:C solu-tion obtained from the first step of the present process has a potency of approxima-tely 10-20 International Units (hereinafter "units") is processed in a column containing aminohexyl substituted agarose. The column is then washed with a buffer solution and the VIII:C is eluted with a calcium ion-containing solution to yield a VIII:C concentration in excess of 1000 units per ml, and being greater than 160,000 fold purified from plasma. Thus, the present method yields unexpectedly high purity procoagulant activity protein in a highly concentrated and therapeutically useful state. Methods used heretofore fail to achieve such no-table results for several reasons. The method of Tuddenham et al, described earlier, employs bound polyclonal antis r instead of the specific and highly selective monoclonal antibodies to VIII :RP as used in the present invention. As a result, fewer specific antibodies to VIII:RP are coupled for a given weight of agarose. In the method of the present invention monoclonal anti-bodies are e~clusively bound to a relatively inert substrate.
When the method of Tuddenham et al is used only 2.6 to 6.4 units of VIII :RP per ml of immuno~lobulin-agarose beads (equivalent to 53.1-82.9% of the amount applied to the column) are removed. This compares to greater than 10~20 units per ml of beads tor 90-100~ of the VIII :RP which is applied to the column) which is recovered when the monoclonal antibody immunoadsorbent of the present inven tion is employed. This ability to adsorb more VIII :C~VIII :RP
(factor VIII~von ~illebrand factor) per ml of beads accordingly ~ 5~

results in a higher concentration of VIII:C when it is subsequentl eluted from the immunoadsorbent. Thus, 10-20 units of VII:C per ml of eluant are obtained with the present invention, in contract to 0.5-1.25 units per ml of eluant with the Tuddenham et al method The present method also permits the selection of a mono-clonal antibody having a high affinity for VIII:RP; however, the use of polyclonal antibodies results in varying affinities. It should be realized that there is an indirect relationship between the affinity of the bound an-tibody for VIII:RP and the elution of VIII:RP. Thus, the higher the aEfinity of the antibody for VIII:~ 7 the less VIII:RP will be present with VIII:C in the eluant. The present invention also makes it possible to produce an unlimited supply of the specified monoclonal antibody, thus eliminating variations among different batches.
Although Austen, as earlier described, has reported the use of aminohexyl-agarose to separate VIII:C from VIII:RP, such a material has not heretofore been used to concentrate VIII:C follow ing a separation and purification step. Heretofore, the highest VIII:C concentrations achieved by using aminohexyl agarose in chromatography were 0.53 units per ml of eluant for human protein and 2.38 per ml of eluant for porcine VIII:C. The present method permits concentrations several orders of magnitude greater than these. Perhaps of even greater significance`, is the fact that t}-e present invention provides for a greater purification of human VIII:C than has ever been reported (164,000 vs 1~,000 fold over plasma). The present me-thod, which is described in more detail hereinafter, yields VIII:C with a specific activity of 2,300 units/mg when commercial concentrate is used. This corresponds ~o a 164,000 fold purification from plasma. The ratio of VIII:C to VIII:RP is greater than 105 as compared to the ratio in plasma.

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DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description provides details of the manner in which the embodiments of the present invention may be made and used in order to achieve the separation, purification and concen-tration of VIII:C to a degree of purity and concentration not known heretofore. This description, while exemplary of the present inven tion, is not to be construed as specifically limiting the invention and such variations which would be within the purview of one skille in this art are to be considered to fall wi~hin the scope of this invention.
A. Preparation of Monoclonal Antibody to VIII:RP
The monoclonal antibody to VIII:RP which is subse~uently bound to the separation substrate may be prepared in a stepwise procedure startin~ with a highly purified preparation of factor VIII/von Willebrand factor (VIII :C/VIII :RP complex~ The purifica-tion is accomplished with material obtained from a plasma source or less highly purified material is used in higher concentration such as commercial extracts available as FACTORATE (trademark o~
Armour Pharmaceutical Co., Tuckahoe, N. Y.~ or Hemophil (trademark of Hyland Laboratories, Costa Mesa, California~. Purification is performed by a standard agarose-gel fil-tration of cryoprecipitate, such as that described by ~immerman and Roberts, "Factor VIII
Related Antigen", appearing in I~,UNOASSAYS: CLINICAL LABORATORY
TECI~NIQUES FOR THE 1980's, R. M. Nakamura et al, eds., Alan R.
Lissr Inc., ~ew ~ork, pp. 339-349 (1980). Mice were injected with highly purified factor VlII/von Willebrand factor obtained from plasma according to the following procedure. On day zero, the mice are injected intraperitoneally with a composition prepared by ~ ~Z5~2 dissolving (or suspending) 10 Mg of the protein in ~.1 ml of buffer containing 0.05 M Tris, 0.15 M sodium chloride, 0.02 sodium azide, 1 mM phenyl methyl sulfonyl fluoride, trasybl 10 units/ml at pH7.3. and shaking with an equal volume of complet Freund's adjuvant. On day 14, the mice are again injected with the same material except that incomplete Freund's adjuvant is sub-stituted for complete Freund's adjuvant. On day 21, the injection of day 14 is repea-ted. On day 38, the mice are injected wi-th purified VIII:C/VIII:RP only. On day 42, -the spleens oE the mice are removed and fused according to a standard procedure, of the type described by J.P. Brown et al "Protein Antigens of Normal and Malignant Eluman Cells Identified by Immunoprecipitation with Monoclonal Antibodies", JOURNAL OF BIOLOGICAL CHEMISTR~, Vol. 225, pp. 498~-4983 (1980~. The standard technique is varied only to th extent that 35% polyethylene glycol 1000 is substituted for 50~
polyethylene glycol. A radioimmunoassay method for clones produc-ing antibody to VIII :RP is performed according to the following procedure. Polyvinyl plates with a "V" bottom, flexible type are coated with 0.1 ml of factor VIII purified from commercial extract according to the procedure indicated above and having a concentra-tion of 0.125 mg/ml of protein. The plates are blocked with albumin, washed with buf~er and incubated with the culture fluids from the clones to be tested. The plates are then washed and re-acted with rabbit anti-mouse IgG antiserum, washed a second time and 125I labeled goat anti-rabbit IgG antiserum is added to the wells and incubated. The plates are again washed, then dried and the wells cut-out and counted. After determining the clones which arQ positive they are subcloned at least twice and stable clones producing antibody to VIII:RP are then injected into the Z51~L5~ 1 peritoneal cavities of Balb/C mice which have been pretreated intraperitoneally with 0.5 ml of pristane at least four days prio to injection of cells. Hybridoma cells are injected at concentra tions of approximately 5 x 1~6 cells per mouse in 0.5 ml of Delbecco's rnodified Eagle's medium without fetal bovine serum. T~ , mice are tapped when bloated and ascites fluid is collected in heparin at approximately 10 units/ml. Ascites fluid from multiple mice is pooled to provide a convenient volume for subsequent isola tion of the monoclonal IgG. If the heparinized ascites fluid is not used immediately, it may be sorted at -70C and thawed just prior to use. The final yield of IgG from the ascites Eluid is approximately lg of I~G per lOOml of ascites fluid.
The specificity of the monoclonal I~G for the purpose of purifying V~ C mav be assessed by coupling the I~G to a separa-tion substrate medium, in the manner described hereinafter, and demonstrating that the bound IgG removes both VIII:RP and VIII:C
from plasma and that the VIII:C may be subsequently eluted with a solution containing calcium ions while the VIII:RP remains com-plexed to the monclonal IgG which is bound to the solid-state substrate.
The monoclonal IgG, which is to be used subsequently to prepare the immunoadsorbent, may be isolated from heparinized pooled ascites fluid il~mediately af-ter collection or a frozen por-tion of the stored solution may be thawed. Regaraless of whether fresh or frozen material is used, the solution is brought to ~C
and treated with an equal volume of prosphate buffered saline solution (PBS), the composition of which is set forth below. The dilut~d ascites is precipitated bv dropwise addition with stirring ~5~

at 4C of an equal volume of saturated ammonium sulfate (SAS);
prepared by boiling an excess of ammonium sulfate in water, coolin to 4C, filtering undissolved crystals and adjusting the pH to 7.0 with ammonium hydroxide. The precipitate and its supernatant liquid are stirred for at least 2 hours and centrifuyed at 4C.
Centrifugations are preferably carried out at 14,000 rpm for 60 minutes (30,000 x 9~. The supernatant solution of ascites is precipitated twice more Wit}l SA~ and the mixture of precipi-ta-te an supernatant liquid s-tirred and centrifuged in the same manner as in the first cycle. The pellets resul-ting from the third precipi-tation are resuspended in a volume o~ PBS equal to that of the diluted ascites fluid and then disalyzed exhaustively against PBS. Clots appearing in the dialysis bags are removed by centri-fugation at 20C. The dialyzed IgG is adsorbed by stirring it with a 5% aqueous solution of aluminum hydroxide at room temperature and centrifuging at 20C after adsorption. The adsorption treat-ent is repeated at least three more times using 2.5~ aluminum hydroxide solution for each treatment after the first. The adsorbed IgG is brought to 4C and reprecipitated once with SAS as aescribed above. The precipitated pellets may be stored at -20C
until used.

. Preparation of the Irnmunoadsorbent The immunoadsorbent is prepared by suitably preparing he monoclonal IgG for coupling, preparing the solid substrate for -oupling and reacting the two components to bind the former to th~
~ater.
(i) Preparation of IgG for Coupling Either freshly precipitated IgG rilay be used or previously ~rozen precipitate may be thawed for use. The material is then 3L25~

dialyzed against PBS, and while still in the PBS, the volume and IgG concentration (A28o/1.4 = mg/ml I~G) are determined. The IgG
is then treated with between 10 and 30 microliters, preferably 20 microliters, of diisopropylfluorophosphate per 50 ml of IgG solu-tion. The resulting solution is stirred at room temper~-ture in a hood or 30 minutes and the treated IgG, immediately prior to use, is dialyzed overn;ght against coupling buffer. The coupling buffe found most suitable is a 0.25M sodium bicarbona-te solution adjuste to a pH of 9, preferably wi-th sodium h~droxide.
(ii) Preparation of Solid Substrate for Coupling A]-though the monoclonal antibody may be bound to any material which does not have a high affinity for protein, particu-larly factor VIII itself, such materials as glass beads, agarose and derivatives thereof are preferred. Most preferred is a cross-linked agarose available commercially as a gel known as Sepharose CL2B (trademark of Pharmacia Fine Chemicals, Piscataway, N.J.).
The method of preparing the preferred immunoadsorbent resin is generally the same as that disclosed in the literature, such as the method of J. Porath et al, JOURNAL OF CHROMATOGRAPHY, Vol. 86, pp. 53-56 tl973). The method found most suitable is as follows: a volume of about 2 liters of Sepharose CL2B is placed in an acid-cleaned 2 liter sintered glass filter funnel. The xesi is washed with water and filtered to a moist cake. The washed resin is placed in a large (approxima-tely 4 liter) glass beaker equipped with a magnetic stirring bar. To the resin is then addcd 750 ml of cold potassium phosphate buffer solution, prepared by mixing one part of a 5M dibasic potassium phosphate solution with , wo parts of 5M tribasic pota~si~m phosphate solution. S~ffici~n e ~' ~5~

cold water is added to bring the final volume to 3 liters. The mixture is then chilled -to 4C ana maintained at between 4-10C in an ice-water bath placed on a magnetic stirring plate. In a hood, cyanogen bromide is added to 300 ml of water in a stoppered glass bottle containing a magnetic stirring bar. The mixture is rapidly stirred until solution results. The cyanogen bromide solution is then added with stirring over a 2 minute period to the cold Sepha-rose mixture. Stirring is continued for an additional 8 minutes and then transferred to a chilled 2 liter sintered glass filter funnel supported in a 4 liter vacuum flas. The cyanogen bromide treated resin is then washed with approximately 20 litèrs of cold water or until the pH of the filtrate is neutral. The washed resi is then quickly equilibrated with cold coupling bu~fer and then transferred to a 4 liter plastic beaker equipped with a large ma~-netic stirring bar.
(iii) Coupling the ~onoclonal Antibody to the Solid Substrat The solid substrate resin, prepared as indicated above, is ready to be used when it is equibrated with coupling buffer and should not be stored thereafter. Accordingly, the resin mixture is combined with the IgG which was previously dialyzed overnight against coupling buffer. The combined resin/IgG suspended mixture is stirred at 4C for a period of about 24 hours. The A28 of an undiluted sample of the supernatant coupling liquid may be deter-mined using bovine serum albumin (BSA) as a standard or Bio-Rad protein assay (Brad~ord reagent) with sSA as standard. The percen _ tage ligand which is coupled may then be calculated. When the above described procedure is followed, this is usually about 95%.
Any remaining active sites on the resin not coupled to antibody m .

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be blocked by washing the resin on a sintered glass filter funnel with cold coupling buffer containin~ 0.lM glycine. The resin is then resuspended in this solution to a final volume equal to that when the resin and ant;body, each in coupling buffer, were combine The suspension is stirred slowly overnight at 4C. The resin is then washed thoroughly with VIII:C-buffer, the composition of which is given below. The coupled, blocked resin is then pre-elut with VIII:C-buffer additionally containing 0.5M calcium ions, pre-ferably calcium chloride. The resin is again washed with VIII:C
buffer alone and stored at 4C or in a continuously pumped column at room temperature until ready for use. The coupling density of IgG to SEPH~ROSE should be 2-5g, preferably 3-4g IgG/liter of SEPHAROSE.
.' C. Separation and Purification of VIII:C
(i) Sample preparation of factor VIII, such as human and animal plasmas and commercial concentrates of factor VIII, may be employed in the present invention and the method is not limited as to a particular type of material. Preferred materials, and those which have demonstrated successful results, are porcine and human plasmas and commercially available concentrates of human actor VIII, such as FACTORATE available from Armour Pharmaceutical Co.
The following description provides details for using both porcine plasma or commercial human concentrate such as FACTORATE:
FACTORATE is reconstituted by adding 25 ml portions of VIII:C-buffer to the contents of each of 20 bottles (25 ml per bottle). The mixture is aajusted to a final volume of 1 liter wit VIII:C-buffer. A sample aliquot of 0.5ml may be removed for assay and the remaining material applied to the immunoadsorbent column overnight at a rate of approximately 60 ml/hour.
Porcine plasma, when not freshly drawn, is citrated by conventional means and stored frozen. When ready to be used i-t is thawed at a temperature of between 35-40C, preferably 37C and applied directly to the column at 60 ml/hour.
It should be noted that while the description of the present invention refers, and is directed primarily, to the use of immunoadsorbent coupled particles in a chromato~raphy column, it is within the scope of this invention to perform batchwise separations by placing the antibody-bound resin particles in a suitable contain , and aEter adding reconstituted concentrate or plasma, VIII:C as outlined above and described in more detail below.
When the process is carried out in a chromatography pro-cess, the following embodiments are preferred:
The resin is placed in a column, such as an Amicon 86001, (trademark of Amicon Corp., Lexington, Mass.), equipped with a peristattic pump and a high flow head. When concentrate is used as the source of factor VIII, for 20 bottles of diluted concentrate approximately 1.5 liters of resin, prepared as indicated above, is used. ~hen porcine plasma is used, 150 ml of resin is used for each liter of plasma.
A~ter the sample is applied to the column, it is washed hith 1 liter of VIII:C-buffer, followed by a second washing with i VlII:C-buffer which additionally contains 0.5M NaCl. Approximately 20 liters of saline-buffer is used when factor VIII is applied as concentrate and 20 bed volumes when porcine plasma is employed.
ptimum results are obtained with a ~low rate of 1 liter/hour.

~ ~L25~15~ 1 Elution of purifiea VIII:C is accomplished with VIII:C-buffer containing calcium ions. Although a linear gradient, as taught by Tuddenbram et al, supra, works well, it is not required in order to accomplish the objeet of this invention; a solution having a fixed calcium ion concentration is quite adequate. Thus, when VIII:C derived from concentrate is being eluted, VIII:C-bu~f r 0.25 to 0.5M with respect to caleium chloride, preferably 0.35M, is used advanta~eously as a ~low rate of between 450 to 750 ml/ho~ r and preferably 600 ml/hour. When the VIII:C is obtained from por eine plasma, elution is perormed with VIII:C-buffer being a calcium chloride concentration of between 0.35 and 0.7~, preferab] Y
0.5M and at a flow rate of between 10 and 30 ml/hour, preferably

2~ ml/hour. Fractions of 12 ml and 3 ml are collected for VIII:C
originating from concentrate and porcine plasma, respectively.
Those fraetions eontaining at least 1.0 unit/ml of VIII:C activit are pooled and the to-tal volume and activity of the pool determin d The VIII:C pool is initially concentrated to 10-20 ml b a stanaard procedure sueh as pressure ultrafiltration. For this purpose, Amicon stirred cell in whieh a YM-10 membrane under 50 p i of nitrogen pressure has been founa to work well. Slow stirring s continued for 30 minutes after nitrogen pressure is released, and the volume and activity o the concentrated pool are determine~
The pool may be stored for a brief period, that is, overnight for example, if a temperature of 4C is maintained.
It may be noted that the immunoadsorbent column describc above may be re~enerated by treatment of the eolumn with 2 bed volumes of 3M aqueous sodium thiocyanate solution run at a flow rate of about 0.5-1 liter/hour to elute ~ ~P.

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D. Concentration of Purified VIII:C
Although t~e VIII:C recovered from the separation from VIII:RP by means of the immunoadsorbent column is highly purified, it is still too dilute to be therapeutically useful. Further con centration is accomplished b~ use of an aminohexyl agarose column which is prepared and used in the followin~ manner:

(i) Preparation and/or Conditioning of a Aminohexyl Agarose- Column:
Aminohexyl agarose is agarose which has been reacted with l,~-diarninohexane to yield an agarose resin having a number ~
6 carbon atom chains, each of which has a -Lerminal amino group. t may be prepared according to the method described by Austen, suprc , or acquired from a commercial supplier. One such material which has been used successfully in the present invention is available under the name of AH-SEPHAROSE 4B (trademark of Pharmacia Fine Chemicals, Piscataway, N.J.).
~ ether prepared or purchased, the resin should be con-ditioned prior to use. This may be accomplished as follows, the volumes, amounts and dimensions being adjusted in proportion to the amount of material to be concentrated:
Approximately 1 gram oE aminohexyl agarose (AH-SEPHAROS
4B) is placed in a sintered ~lass ilter funnel and washed with a least 200 ml of 0.5M sodium chloride, while stirring. The resin is then equilibrated with VIII:C-buffer and packed in a column of approximately 0.9 cm diameter. A Bio-Rad Econo-Column with flow adapters has proven quite suitable for the type of use considered here. The bed volume of the packed column is appro~imately 4 ml.

(ii) Application to and Use of the Aminohexyl Agarose Column The concentrated pool, prepared as described a~ove, is diluted 1:10 in VIII:C-buffer to a final concentra~ion of 100-200 ~ ~S ~ 2 ml when usin~ the amounts of resin and column size as described in the immed;ately preceding section. The diluted pool is applied to the column at a flow rate of 200 ml/hour.
The column is then washed with VIII:C-buffer which con-tains calcium ions, preferably from calcium chloride. The solu-tion should be between O.OlM to 0.03M, preferably 0.025M with respect to calcium ions.
Elution of the concentrated VIII:C is achieved at a flow rate of between 5 to 20 ml/hour, preerably 10 ml/hour with VIII:C-buffer containing a higher concentration of calcium ions than was employed with the preceding washing step. Again, calcium chloride is the preferred source of calcium ions in a concentra-tion of between 0.25 to 0.5M, preferably 0.3M. Fractions of 1 ml volume are collected and assayed as described below. Collected fractions may be sorted at 4C or frozen. Preparations of VIII:C
obtained from a porcine plasma source whould be stabili~ed with 1 human serum albumin prior to storage.
Assays may be performed by ailuting the fractions with VIII-C-buffer if necessary and further diluting the fraction 1:100 in assay buffer prior to addition to the substrate.
standard partial thromboplastin time assay is employed.
The composition of the buffer solutions is as follows:
Phosphate Buffered Saline Solution:
1.6g sodium phosphate, monobasic monohydrate 8.4g sodium phosphate, dibasic anhydrous 61.4 sodium chloriae Water to 7 liters p~ of buffer is 7.2 1251:L5Z

VIII:C-Buffer ml 0.02M imidazole ml 0.15M sodium chloride ml O.lOM lysine ml 0.02% sodium azide pH of buffer is adjusted with concentrated hydrochloric acid to 6.8.
The data listed hereinafter in Tables I and II are repre-sentative of that ob-tained according to the present inven-tion, as escribed above.

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Ul . ~ ¦ ~-- h ,¦ -- ¦ c o ` ~ 2 ~1 Although only preferred embodiments are specifically illustrated and described herein, it will be appreciate that many modifications and variations of the present invention are possible in light of the above teachings and within the purview of the appended claims without departing from the spirit and in-tended scope of the invention.

Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A monoclonal antibody for isolation of VIII:C from VIII:C/VIII:RP, said monoclonal antibody being specific to VIII:RP and remaining complexed to VIII:RP when subjected to a saline solution elution.

2. A monoclonal antibody of claim 1, wherein said saline solution contains calcium ions.

3. A monoclonal antibody of claim 2, wherein said solution is calcium chloride.

4. A monoclonal antibody of claim 3, wherein the concentration of said calcium chloride is from 0.25M to about 0.5M.

5. A monoclonal antibody of claim 1, 2 or 3, wherein the monoclonal antibody is freshly precipitated.

6. A monoclonal antibody of claim 4, wherein the monoclonal antibody is freshly precipitated.

7. A monoclonal antibody of claim 1, 2 or 3, wherein the monoclonal antibody is frozen precipitate.

8. A monoclonal antibody of claim 4, wherein the monoclonal antibody is frozen precipitate.