Pasteurized, isoagglutinin-free factor VIII preparation and a process for its production

A process for the production of a preparation of blood coagulation factor VIII which makes it possible to obtain a pasteurized product which is virtually free of immunoglobulins, isoagglutinins, fibronectin and coagulable fibrinogen is described. A product of this type can be used for the treatment of blood coagulation disturbances.

The invention relates to a process for the production of a pasteurized and 
purified preparation of blood coagulation factor VIII. This preparation 
can be used for the treatment of blood coagulation disturbances. 
Blood coagulation is a complex process which takes place via several 
reaction steps and which involves at least 13 coagulation factors (F) 
which are identified by roman numerals. The coagulation factors are 
predominantly proteins and, in particular, proteins equipped with the 
properties of proteases or accelerators. The only actual substrate is 
fibrinogen which, in cases of injury, is converted by thrombin into its 
insoluble form, fibrin, which forms the primary wound closure. If one of 
the 13 coagulation factors is missing, the formation of thrombin and 
fibrin does not take place; the consequence is hemorrhage. An instance of 
this is hemophilia A which is the most widespread disease with a tendency 
to hemorrhage and which is due to a deficiency of factor VIII. Both 
hemophilia A and B (F IX deficiency) can only be effectively treated by 
replacement of the factor which is lacking. 
The problem of obtaining F VIII from human plasma in good yield and high 
purity, as is necessary, in particular, for the self-treatment of 
patients, still has no optimal solution. This particularly applies to 
pasteurized F VIII concentrates which have increasingly displaced the 
conventional commercial product because it has been possible with them to 
eliminate the risk of transmission of hepatitis. 
The isolation of a highly purified F VIII in good yield is made difficult 
by, in particular, the fact that although F VIII is enriched in the 
cryoprecipitate, it is associated with fibrinogen and fibronectin, which 
are two proteins which have relatively high molecular weights and are 
sparingly soluble and have similar physicochemical properties to F VIII. 
A factor VIII concentrate should be as pure as possible, that is to say 
free of undesired concomitant proteins and, in particular, of 
immunoglobulins, including isoagglutinins; this is because there are 
indications that administration of non-specific proteins leads to 
overstrain of the reticuloendothelial system (RES) and to impairment of 
the immune defenses manifested by a change in the composition of the 
lymphocyte population and of the immunoglobulins. The significance of this 
becomes more evident when connected with the fact that hemophiliacs have 
to undergo life-long treatment with such F VIII concentrates. This results 
in the demand for a native, highly purified, pasteurized F VIII 
concentrate, i.e. a product which rules out transmission of hepatitis 
viruses and other infectious material and rules out sensitization to 
isoantigens. 
This invention relates to a product of this type which rules out the 
transmission of hepatitis and is free of isoagglutinins, and to a process 
for its production. 
It has been found that a solution containing F VIII but which is virtually 
free of factors of the prothrombin complex (F II, VII, IX and X) can be 
pasteurized in a manner known per se after the addition of stabilizers to 
protect against thermal inactivation, the heated solution can be treated 
with an anion exchanger in the pH range 5-6.5, and the adsorbed F VIII can 
be washed free of concomitant proteins, especially fibrinogen, fibronectin 
and immunoglobulins, including isoagglutinins, eluted with a concentrated 
solution of Na, K or Ca salts with a halogen, and obtained by, for 
example, precipitation from the eluate. 
Ecteola-cellulose and an anion exchanger bearing QAE (quaternary 
aminoethyl) groups have already been used for the purification of F VIII 
(van Creveld, S. et al., Thromb. Diath. Haem. (1961) VI, No. 2/3, 282 and 
Baugh, R. et al., Bioch. Biophys. Acta (1974) 371. 360). However, transfer 
to pilot-plant or manufacturing scale has not been possible. It has merely 
been possible to use ion exchange chromatography for the final 
purification of prefractionated material (Fay, Ph. J. et al., Proc. Natl. 
Acad. Sci. (1982) 79, 7200). Adsorption with high specificity takes place 
only at a pH around 5.5; however, at this most of the proteins contained 
in the cryoprecipitate, in particular human fibrinogen, precipitate out, 
especially on contact with the adsorbent in the column. Moreover, 
according to the papers quoted, relatively large amounts of adsorbent are 
necessary. This in turn appears to have had an enormous effect on the 
yield, which was tiny, apparently owing to non-specific adsorption of F 
VIII--its physiological task is, after all, to adhere to non-physiological 
surfaces--to the large amounts of adsorbent. It was also found that the 
material finally purified on a QAE exchanger rapidly lost activity; for 
this reason, anion exchangers have not been used for the manufacture of F 
VIII. 
However, it has now been found, surprisingly, that chromatography on basic 
ion exchangers is perfectly suitable for the manufacture of F VIII 
preparations. It was surprising that, starting from a pasteurized 
cryoprecipitate, it is possible to obtain a highly purified F VIII 
preparation in one step by using anion exchange chromatography. 
Thus the invention relates to a process for the production of a factor VIII 
preparation, in which a solution containing factor VIII is, in the 
presence of stabilizers, pasteurized and purified, which process comprises 
the solution being treated with an anion exchanger which is based on 
carbohydrates, the exchanger being washed, and the F VIII being eluted. 
Suitable starting materials are a cryoprecipitate obtained by the method of 
Pool et al., Nature (1964) 203 312, Cohn fraction I (Minot, G. R. et al. 
J. Clin. I -est. (1945) 24, 7047, plasma, F VIII:C-containing cell culture 
medium and side fractions containing F VIII obtained from the latter. 
However, it is advantageous to use directly cryoprecipitate or Cohn I 
fraction. 
It is possible to use as the stabilizer, for protection against thermal 
inactivation of F VIII during pasteurization, carbohydrates and amino 
acids, preferably 35-60 g of sucrose per 100 g of solution and 1-3 mol of 
glycine per liter of solution and, where appropriate, calcium ions. The 
method can be that of, for example, German Offenlegungsschrift 2,916,711 
or 3,237,512. 
Examples of suitable anion exchangers for the adsorption of F VIII are 
exchangers bearing DEAE, QAE or ecteola groups, specifically those based 
on, in particular, cellulose, sephadex or sepharose, but DEAE-sepharose is 
preferred. 
In contradistinction to van Creveld and Baugh (see above) these exchangers 
are in fact suitable for purification of F VIII, but under conditions 
which have not hitherto been disclosed and are described below. 
The adsorption conditions have proved to be vital. This is because it has 
been found, likewise surprisingly, that in a physiological saline medium, 
preferably at pH 5.5, F VIII is substantially selectively bound to 
DEAE-sepharose, while concomitant proteins, such as fibrinogen and 
fibronectin, remain in the supernatant (batch process) or pass through a 
column. Finally, it has been found, again surprisingly, that a pasteurized 
solution of the cryoglobulins can in fact be chromatographed at pH 5.5 in 
a physiological saline medium, since certain cryoglobulins precipitate 
under these conditions, especially fibrinogen. The adsorption and, in 
particular, the specificity with which F VIII is bound to the anion 
exchangers decrease greatly as neutrality is approached. However, under 
normal chromatography conditions, DEAE is only loaded when the pH exceeds 
7.0. Nevertheless, precipitation does not take place in the process 
described here, because the carbohydrates which have been present in the 
cryosolutions since the pasteurization keep the fibrinogen and cig in 
solution in a slightly acid medium. 
Thus an advantage of the process described is that on adsorption of 
pasteurized cryoglobulin onto the anion exchanger the fibrinogen and 
fibronectin remain in the supernatant or effluent and can be obtained from 
them as pasteurized products. An example of a method for fibronectin is in 
German Offenlegungsschrift 2,848,529. 
The chromatography on, for example, DEAE- or QAE-exchangers is carried out 
in a slightly acid medium (pH 5.5) and on exchangers which have been 
appropriately equilibrated, for example with 0.1 mol/l Na acetate buffer 
containing 0.1 mol/l lysine. It is also possible to use this buffer to 
dilute the pasteurized solution containing F VIII to twice the volume 
before it is treated with the exchanger in a batch or column process. 
Batch adsorption, which is preferably used, has the advantage that it is 
possible to follow the binding of F VIII to the exchanger during 
adsorption by functional determination of F VIII in the supernatant, and, 
after the activity has disappeared, it is possible to separate the 
nonadsorbed factors from the ion exchanger by sedimentation or 
centrifugation and, immediately thereafter, to start with the washing of 
the exchanger loaded with F VIII. Whereas the washing is advantageously 
carried out as a batch process, for example on a suction filter, it is 
advisable to transfer the exchanger to a column for the solution since 
elution by column chromatography has the advantage that the F VIII is 
obtained in a relatively concentrated form--of the order of 50-100 IU F 
VIII/ml under experimental conditions. 
The buffers which are suitable for washing the exchanger which is loaded 
with F VIII are, in particular, those which, on the one hand, do not 
dissolve off the F VIII but, on the other hand, are suitable for removal, 
which is as nearly quantitative as possible, of nonspecific proteins, such 
as the immunoglobulins, and thus also the isoagglutinins. Surprisingly, an 
appropriate buffer has proved to be a buffer in which the starting 
material can be dissolved for the adsorption and which contains 0.1 mol/l 
Na acetate, 0.1 mol/l lysine and 1 g/l NaCl at pH 5.5. The ion exchanger 
loaded with F VIII was washed with this buffer until the eluate was free 
of isoagglutinins. The highly sensitive Coombs test was used for testing, 
this also indicating incomplete antibodies. 
Concentrated salt solutions, for example those containing NaCl, are 
suitable for the desorption of the F VIII from the anion exchangers. 
However, other salts of halogens with Na, K or Ca have proved to be more 
advantageous: KBr, NaBr and CaCl.sub.2. They have the advantage that the F 
VIII is eluted as a relatively sharp peak with a high activity per unit 
volume, and this is an important advantage for the precipitation. 
The concentrations are in the range 0.05 mol/l up to the saturation limit. 
Finally, the yield also depends quite significantly on the rate of elution, 
which should be of the order of 1-10 ml/cm.sup.2 /h, preferably 5-7 
ml/cm.sup.2 /h. 
The eluate containing F VIII can be concentrated by the customary methods, 
that is to say by precipitation with neutral salts, such as ammonium 
sulfate or NaCl, advantageously with NaCl which can be dialyzed out more 
rapidly than ammonium sulfate and, moreover, need not be removed 
completely. 
The further processing of the precipitate containing F VIII is carried out 
in such a manner that the residue from the precipitation is dissolved in, 
for example, a buffer of pH 6.9 which contains Na citrate (0.02 mol/l), 
NaCl (0.06 mol/l), glycine (20 g/l) and albumin (5 g/l) (dialysis buffer) 
and is dialyzed to equilibrium against the same buffer without albumin. 
The dialyzed solution is diluted with the albumin-containing dialysis 
buffer to an activity of 25 to 30 IU F VIII/ml and, after sterilization by 
filtration, is dispensed into containers and, where appropriate, 
freeze-dried. The final product is a white lyophilizate which dissolves in 
less than one minute, has a F VIII activity of 5-10 IU/mg protein, and is 
pasteurized and free of isoagglutinins. Compared with products of the 
state of the art, it has the advantage that it contains no undesired 
proteins, in particular those which, as isoantigens, might lead to 
sensitization. The product has not given rise to blood-group 
incompatibilities. Transmission of viral diseases, in particular the 
various types of hepatitis, appears to be ruled out. The advantages of the 
process include the facts that it is relatively straightforward and thus 
can be transferred without difficulty to an industrial scale and that it 
comprises few working steps. The small number of working steps is 
reflected by the good yield, since experience has shown that every 
purification step is associated with loss of activity to a greater or 
lesser extent. 
Thus the invention also relates to a highly purified, pasteurized factor 
VIII preparation which is virtually free of immunoglobulins, 
isoagglutinins, fibronectin and coagulable fibrinogen and which has a 
specific clotting (C) activity (F VIII:C) of about 100 U/mg protein and a 
ratio of F VIII:C to F VIII R:Ag (related antigen) of &gt;1. 
F VIII can be determined by the following procedure: One part, for example 
0.1 ml of partial thromboplastin, for example that prepared by the method 
of German Offenlegungsschrift 2,316,430, is mixed with one part of F 
VIII-deficient plasma and one part of diluted normal plasma. This mixture 
is maintained at 37.degree. C. for 6 minutes. After the addition of one 
part of a 0.025 molar calcium chloride solution prewarmed to 37.degree. 
C., the time which elapses between addition of the calcium chloride 
solution and the appearance of a clot is determined. A calibration curve 
drawn up using serial dilutions of normal plasma is used for quantitation. 
1 international unit (=1 IU) of F VIII is equivalent to the F VIII activity 
of 1 ml of normal plasma as a substandard to the 3rd International WHO 
Standard. 
The process for obtaining a pasteurized F VIII preparation which is free of 
isoagglutinins is illustrated below:

EXAMPLE 
1. Starting material 
250 g of crude cryoprecipitate were dissolved by heating at 
30.degree.-37.degree. C. in 750 ml of an NaCl solution (0.08 mol/l), which 
contained glycine (0.25 mol/l) and heparin (1.25 USP-U/ml). This resulted 
in 1,000 ml of a solution with a concentration of 0.06 mol/l NaCl, 0.2 
mol/l glycine and about 1 USP-U heparin/ml. The pH of the solution was 
adjusted with 1N HCl to 6.5. 
2. Aluminium hydroxide adsorption 
80 ml of a suspension containing 10 g/l aluminium hydroxide (Behringwerke 
Marburg) were added to 1,000 ml of solution from 1. and the mixture was 
stirred for 15 minutes (temperature about 30.degree. C.). It was then 
centrifuged at 3,000.times.g for 15 min, the residue was discarded, and 
the supernatant was pasteurized after the addition of stabilizers. 
3. Pasteurization 
The following stabilizers were added, in this sequence, to 1,000 ml of 
supernatant from 2: 
5 ml of CaCl.sub.2 solution, 1 mol/l (5 mmol/l) 
1,000 g of sucrose (500 g/kg solution) 
150 g of glycine (2 mol in 1 l of solution). 
The pH was adjusted with 2N NaOH to 7.3. The volume increased to 1,700 ml 
owing to the additions. The solution was kept at 60.degree. C. in a water 
bath for 10 hours. 
4. Ion exchanger treatment 
1,700 ml of solution from 3. were diluted with 1,700 ml of a solution which 
contained 0.2 mol/l Na acetate, pH 5.5, and 0.2 mol/l lysine. The pH was 
adjusted with dilute acetic acid to 5.5, and 70 ml of DEAE-sepharose 6 B 
Cl, equilibrated with a solution containing Na acetate (0.1 mol/l), pH 
5.5, lysine (0.1 mol/l) and NaCl (1 g/l), were added. The mixture was 
stirred at room temperature for 2-3 hours, and the binding of the F VIII 
was determined by determination of the activity in the supernatant. When 
the F VIII activity had decreased from 4 IU to 0.1 IU/ml, the adsorbent 
was removed by centrifugation. 
The supernatant was poured off and the adsorbent was separated from the 
remainder of the supernatent. Entrapped protein was removed from the 
sepharose by washing with a solution containing 0.1 mol/l Na acetate, pH 
5.5, 0.1 mol/l lysine and 1 g/l NaCl, and it was then transferred as a 
slurry in the same buffer to a column (dimensions: 10.times.3 cm). 
The exchanger was washed in the column until there was no longer any 
measurable absorption of light at 280 nm and the isoagglutinin values in 
the Coombs test were at the detection limit. 
5. Elution 
The exchanger from 4. was eluted with a solution, of pH 5.5, containing 0.1 
mol/l Na acetate, 0.1 mol/l lysine and 0.3 mol/l CaCl.sub.2. This resulted 
in the appearance of a peak measurable at a wavelength of 280 nm. The 
corresponding fractions were collected, and a volume of 180 ml containing 
40 IU F VIII/ml was obtained. 
6. Precipitation of F VIII 
2.2 mol/l glycine and 150 g/l NaCl were added to 180 ml of eluate from 5., 
and the mixture was stirred at room temperature for 30 min. 
The precipitation was recognizable by a marked opalescence. The precipitate 
was separated off by centrifugation at 30,000.times.g in an 
ultracentrifuge for 30 min and, after the supernatant had been poured off, 
it was kept at 4.degree. C. overnight. 
7. Working up 
The precipitate from 6. was taken up in 145 ml of buffer, of pH 7.0, which 
contained 0.02 mol/l tri-Na citrate, 0.06 mol/l NaCl, 10 g/l glycine and 5 
g/l human albumin (dissolving buffer). The activity determined in the 
solution was 40 IU F VIII:C/ml. It was dialyzed against the abovementioned 
buffer containing no albumin, at room temperature for 3 hours, and the 
dialyzate was warmed to 30.degree. C. and centrifuged at 30,000.times.g 
and 25.degree. C. for 30 minutes. After determination of F VIII, which 
showed 36 IU/ml, the solution was adjusted to 30 IU/ml with dissolving 
buffer, and the solution was warmed to 37.degree. C. and sterilized by 
filtration through a membrane filter. 
It was dispensed into vials, frozen and freeze-dried.