CA1198366A - Antihemophilic factor concentrate - Google Patents

Abstract

Abstract of the Disclosure Novel antihemophilic factor concentrates and process for making the same are disclosed. An aqueous solution of antihemophilic factor proteins is obtained, preferably by solubilizing antihemophilic factor proteins from blood plasma cryoprecipitate in water. The aqueous solution is purified to remove unwanted protein, fox example, by mixing the aqueous solution with aluminum hydroxide. Then, the aqueous solution is ultrafiltered to concentrate it, mixed with buffer and saline and adjusted to an acid pH, and freeze-dried.

Description

/r j i6 Background of the Invention Field of the Invention: ~his invention relates to and has among its objects the pro~ision o novel an~
hemophilic ~actor concentrat~s and methods for making them.
Further objects o~ the invention will be evident from the following description wherein parts and percentages are by weight unl~ss specified otherwise.
- Description of the Prior ~rt. Currently, a~ti hemophilic factor ~AHF),otherwise ~nown as Factor VIII, is prepared from human plasm~. Cryoprecipitate is recovered from thawed pools of fresh rozen h~an pla3ma by centri-fugation and diced and washed to remove soluble proteins.
Then, the cryoprecipitate is extracted or solubilized with water. The p~ of the aqueous solution iB adjusted ~o slightly acid and the ~olution i~ chilled to ~eparate extraneou~ non-~HE' pxotein. Next, the aqueous ~olution is treated with aluminum hydroxide to further remove unwanted protein, particularly prothrombin compl0x proteins. This purification technique u~ing aluminum hydroxide has been described by ~ershgold et al in J. Lab. & Clin. Med., 1966, Vol. 67, pages 23-32, by Mozen at the Twenty-Fourth ~nnual Wayne State University S~mposium on Blood, Detr~it, Michigan ~Ja~uary 1976) and at the XV Congxe~s of the International Society of HematolQgy, Jerusalem, Israel (1974), and by Liu et al, U.S. Patent No. 4,170,639 (October 1979). Salt and a buffer are added to the aqueous solution which is then freeze-dried after adjustme~t of the pH of the aqueou~
~olu ion to slightly acid.
In large sca~ P processing, it i~ d~sirable to remove water from ~he aqueous ~BF ~olution prior to free~e-drying it. To this end the AHF concentrate is reprecipitat~d before it is lyophilized. The aqueou~ ~olution is treated with ei~her cold ethanol,polyethylene glycol, ox glycine which result~ in precipitation of ~HF protein~ The ,l pre~ipitat~ i~ collected by cen~rifugatiQnl mixed with water buf~er, ~ n~ and acid, and freeze~dried.
Li~ in ~S. Patent No. 4,170,639, discloses that an aqueous A~F ~x~ract, aftex purification with aluminum hydroxide and reconstitution with buffer and saline and adjustment to an acidic pH, may be subjected to ultra-filtration to concentrate it prior to freeze-drying. The ultrafiltration is conducted only on reconstituted AHF
extract that has been adjusted to an acidic pH using a membrane with a molecular weight cut~off of one million daltons.

Summary of the Invention It has now been di~covered that water can be removed effectively from aqueous solutions of AHF proteins by subjecting an P~ solution to removal o water, prefer-ably by ultrafiltration, after puriying it to remove unwanted proteins but prior to mixing it with buffer and saline. Studies have shown thak water removal is achieved in -the process of the invention without signiflcant change in the activity of the ~HF proteins. The ultrafiltration technique employed in the prior art processes causes a re-duction in yield of ~HF activity, which the present method unexpectedly avoids.
The mixing with buffer and saline is suitably followed by adjustment to an acidic pH.

~r~

A primary advan-tage of the present invention is that it results in essentially complete recovery of A~IF
activity. The conventional reprecipitation and ultra-filtration methods result in incomplete recoveries of Factor VIII activity, presumably due to denaturation of the AHF proteins. No significant reduction in AHF acti-vity was observed in our process wherein ultrafiltration was applied to the aqueous e~tract prior to mixing with buffer and saline and pH adjustment for a period of at least five hours. The ultrafiltration process of the prior art results in abouta 15% or more loss of anti-hemophilic factor.
Still another advantage of the present invention is the significantly hiyher content of Von Willebrandt factor (VIIIR:WF) in the product of our process. It is possible to obtain a product containing substan-tially the same proportions of VIIIR:WF and procoagulant factor (VIII:C). Such a product approaches more closely the native VIII:C state and should ~e suitable for Von Wille-brandt patients, i.e., patients suffering from Von Wille-brandt's disease~ In addition, the in ivo half life of this product is increased. In general, the VIII:C/
VIIIR:WF ratio in the products of the invention is within the range of a~out 1/0.5-1 and usually about 1/1, whereas in the alcohol precipitation method the above ratio is about 1/0.1-0.4, usually 1/0.2. The concentration of VIIIR:WF in the instant products ~s ~reater than 15 units pPr milliliter (ml), generally about 15-30 units per ml and sometimes as high as lQ0 units per ml. ~he concentrations of VIIIR:WF obtained with the alcohol precipitation step of concentration ~or example, ~enerally are less than 15 units/ml, i.e.j about 5-10 units/ml.
Another advantage of the i~vention is the improved ratio o ~HF prote~ns to milligxams of fibrinogen~ The increased yield of AHF concentrate over that obtained using conventional r precipitation results ~n a signi~icant decrease in the amount of fibrinogen per unit dose. Thu~, ~ibrinogen overload in patients receiving laxge volu~es of ~F concentrat~s may be avoided by using the products of our invention. The ratio of AHF proteins to milligrams of fibrinogen (VIII:C/mgO 0) of the produc~s of the invention ~alls within the range of about 2.6-4.0/1, usually about 3.0/1. In the alcohol reprecipi- ;
tation method of concentration used by the art, the VIII:C/
mg 0 ratio of the ~inal products is about 1.0-2.5/1, generally about 1.5/1. The pre~ent products haviny improved VIII:C/
VIIIR:WF and VIII:C~mg ~ heretof~re have been unavailable.

De~cription of the Pr~ferred Embodiments As mentioned aboveO ~HF con¢entrate is obtained from human plasma. I~ general, thP preparation of AHF concentrate is carried out by modification and ref~ne~nt of a method irst described by ~ershgold et al, J. Lab. Clin. Med., ~upra.
Cryoprecipitate i8 removed ~rom pools of fresh frozen human plasma, which have been thawed at les5 than 5 C~ The cryoprecipitate is diced and washed with bufex and then fiolubilized in water. The pH of the aqueous ~HF ~olutio~ is adjusted preferably to within the range of 6~40-6.95 by addition of a biologically acceptable acid, particular acids of this ~ype being well known in th2 art. ~ny precipitate that forms after chilling the solution at less than 10 CO is remo~ed by decantation ~r centrifugation. The aqueous 801ution cont~;ning ~he active ~HF c ~o~ent& is mixed with alumin~m hydroxide to ~electively xemove unwanted proteins.

~ 3 --~1 It is a characteristic ~f this treatment that the pH
remains v~rtually constant throughout. As a result of the aluminum hydrox~de treatment unwanted protein is selectively xemoved from ~he aqueous AHF solution without substantial loss of AHF potency. It is this purified aqueous solution to which the process of our invention is applied.
In accordance with the invention aqueous AHF
solution processed as above is subjected to uItrafiltration.
The solution is contacted with a particular semi permeable membrane until the desired amount of water is removed, namely, that amount o~ water which, when xemoved, gxeatly facilitates the lyophiliza~ion o~ the final solution. ~lembranes suitable for the process of our invention ~hould have a nominal molecular weight cut-off less than one million daltons, preferably within the range of 10,000 to 300,000 daltons.
~embranes with moleculax weight cut-offs of one million daltons physically entrap the AHF proteins and, thus, the yield of AHF concentrate i~ reduced. Typical ultrailtration membra~es that may be used in the invention (with corxesponding nominal molecular weight cut~offs) are Amicon XM50 (50,000 daltons, manufactured by Amicon Corporation, Lexington, Mass.), ~micon PM10 (10,000 daltons), ~micon ~1lOOA ~100,000 daltons), Amicon XM300 (300,000 daltons), and the like. In a preferred embodiment of the invention ultrafiltration of the aqueous A~F ~olution is conducted with hollow iber~ in an ultrafiltxa tion unit such as for xamplel the ~micon DC 30 (30 sq. ft.
filtration area) hollow fiber unit, using an ultrafiltration membrane such as the ~micon ~lOP10 (10~000 daltons~ cartridge, or the Pqui~alent.
It is preferred in carrying out the method of he invention that the l~min~r flow and shear rates at the ultra-$iltration membrane wall be lowO Excellent results are achieved with lpr;n~r flow rates at he wall l~ss than Reynolds Number 2000, preerably within the range o~ Reynolds N~mber 200 to 300, and shear rates at the wall less than ,1 1000 rever~e ~econds (sec 1~, pre~erably within the range of 200 ~o 300 sec 1. It is to be realized, of Gour~e ~ that higher flow rate~ and the resulting sheax at the membrane wa~l during ultrafiltra-ion of the aqueous solution in accordance with the inYenti~n will yield a concentrated AHP produc~ in a shorter processing time~ ~vwever, the activity of the AHF concentrat9 i5 reduced at these higher rates due to AHF protein ~ ~ r d ~ r l~

~'1 denaturation. In this respect, also, special care must be taken to min;m;ze air uptake and other interfacial effects in a shear field such as foaming and the like in order to ~; m; ze denaturation of the AHF proteins.
The t~pe of recirculation pump employed during the ultrafiltration procedure is an important aspect of the present invention. Diaphragm pumps (generally air-operated]
yield ~in;~l denaturation ~f the AHF proteins whereas centrifugal pumps are ~ndesirable because of excessive loss in AHF protein acitivity that results during their u~e.
Suitable pumps to be used during ~he ultrafiltr~tion step are, by way of example, Amicon LP-20 (Amicon Corporation) air pre~sure operated diaphragm pump and the Warren Rupp 5andpiper pump (Model SA1-A-DB l-SS) (Thomas and Associates, Corte Madera,' Caliornia).
Following ultrafiltration of the aqueous A~IF
solution the ~olution i~ mixsd with buffer and ~aline ~ is conventional in the axt. To this end aqueous s~dium chloride i~ added to.the aqueous extract in ~iologically acceptable amounts usually to a level of 0.05-0.30 molar, preferably 0.15 molar. Furthermore, an appropriate biologically-acceptable buffer~ such as sodium citrate, is added thereto to a l~vel of 0.005-0.03 molar, preferably 0.01 molar. If necessary, the pH o the aqueous extract i~ adjusted to within the range 6 . 4 to 7.4 (established by regulation by the Food and Drug i~tlm; n i stration) by addition of a biologically acceptable acid. The aqueous AHF solution is filtered to ~~ ~ve particles and then sterile iltered.
$t i5 important to note that the ultrafiltration procedure for re~oving water from the aqueous A~F solution must be applied to the solution prior to mixing thP solution with bu~fer and s~l;ne. If not, the benefits and advantayes enum~rated above are not r0alized, particularly with respect to yield of ~HF produot. I
Following sterile ~iltration o~ the so-treated a~ueous ~F solution, the solution i~ free~e-dried llyophilized~. The ~olution m~y be aseptically filled into containers of an appropriate ~iz~ to be quick-frozen and the frozen material lyDphili~ed under high vacuum as is ,1 36~

well known in the art. The containers with freeze-dried product therein are sealed, and the product is ~tored at a temperature of about 2-8 C. until it is used.
For infusion, the contents of each container are reconstituted in sterile distilled water yielding a solutioII containing approximately 25 AHF activity units per milliliter.
It is within the purview o the invention to follow the above-described ultrafiltration procedure with a ylycine precipitation step to improve the color and i clarity of reconsti uted final freeze-dried product. To ! this end the ultrafiltex d aqueous solution is mixed with glycine to a concentration therein ~bout 1.6-2.2 molar, preferably 1D 9 molar, at a temperature of about 5-20 C.
Optionally, the mixture can be mixed also with sodium citrate and saline to concentrations of 0.005-0.03 molar and 0.05-0.30 molar, r~spectively. The mixture is held for about 30 minutes or more, preferably 30-120 minutes, and optimally ~or 60 minutes. When the ~reeze-dri~d product, prepared in accordance with the above teaching, is reconstituted, it has a pale yellow color and a clarity grPater than 80~.
It is noteworthy that the glycine precipitation Btep described ahove must be ~pplied to ultrafiltered AHF
~olution~ I~ the ultxafiltration step i~ omitted appxoximately one half o~ the ~HF activity i8 lost in the final product.
Furthermore, in order that the glycine treatment be successful and that A~F activity 105s be avoided, the a~ueous AHF solution must contain at lea~t 50 milligrams of protein p~r ml. prior to treatment with ~lycine.
~ he precipitate that ~orms as a result of the glycine treatment i~ separated from the A~IF solution by con~entional m~ans such as centrifugation, ~iltration, and the like, and the p~ecipitate i~ dis501ved in buff@r and ~ e a~ de~cribed herei~hove. After p~
adju~tme~t a6 above, the ~queou~ A~F solution is freeze-dried ~o yield a dri~d ~F concentrate.
It ~h~uld be o~iou that the ultrafiltered AHF
~oluti~n wi~h a redu~ed water conten~ alRo may be considered to be ~n AHF co~c~ntrate, thsugh not a completely dry one.

~ ., 6~

Examples The ~nvent~on i~ demonstrated ~urther by the following illustrat~ve examples.
In the examples total protein was determined by absorbance measurements at 280 nanometers.
Procoagulant activ~ty (~ C) was assayed by one stage Acti~ated Paxtial Thromboplastin Time (APTT) test modified from the methods o~ ~angdell -et al, ~. Lab. Clin.
Med., ~ol. 41, pages 637-647 (1953) and Proctor et al, Am. J. Clin. Path., Vol. 36~ page ~12 (1961).
Ristocetin-Willebrandt factor activity tVIIIR:WF) was as~ayed with gel-iltered platelets accordillg to the method of 0150n et al, Am. J. Clin. Path., Vol. 63, pages ~10-~18 (1~75).
Qua~titative factor VIII antigen (VII~R:Ag) determinations were done according to the procedure of Laurell, Anal. Biochem.~ Vol. :15, pages 45~5~ (1966).
Antiserum against the ~actor VIII related p.rotein~
was obtained ~rom ~ehring Diagnostics ~ommerville, New Jersey).
Protein species distribution was assayed by cellulose ac~tate electrophoresis.

Ex~mple 1 Production of Agueous Svlution o~
AH~ Proteins A modified method of Hershgold t al~ , was followed. Fresh ~rozen human plasma was thawed at not more than 5 C. and ~armed to not more than 15 C. The so~warmed pl~sma was chilled to 2~ C. After 3 hours the ~nsoluble cryoprecipitate was collected by centrifugation at not more than 10 C.
~ he cryopxecipitate ~1 kg.~ was diced and ~u~pended in 10 1. of 3terile water at 32 C. fox not more than 2 hours. Then, the mixtur~ was adjusted to p~ 6.8 by addition of 0.1 N hydrochloric acid and chilled to 5 C. Precipitate ~as l~- -,v~d by ce~trifugation at 5~ C.
The aqueou~ solu~ion ~upernatant3 wa~ mixed with a 3~ ~u~pension of aluminum hydroxide i~ water in the ra io of 0.1 g of alumi~um hydroxide per 1 ~ of protein. The -- 7 ~

~3~

mixture was ~tirr~d for 30 min, at 5 C. and the aluminum hydroxide was removed by filtration and centrifugation.

Example 2 Ultrafiltration of AHF Solution in Thin Channels The aqueous solution 1300 ml,) from Bxample 1 was ultrafiltered through a variety of thin channel ultra-filtration membranes in an Amicon TC~-10 ~hin-channel system at the specified temperature un~il concentrated to a volume of 50 ml. The membranes employed were the Amicon XM50, Amico~ XMlOOA, and ~micon ~M300 (all Amicon Corporation), ~illipore PSVP (106 daltons~ Millipore Corporation, Bedord, ~lass.~. ~he ultrafiltered material wa~ analyzed by ~he above-described method.
~ he results are su~narized in Table 1.

Example 3 Ultxafiltration o$ ~HF ~olution in Hollow Fibers Pilot Scale Runs Aqueous AH~ .~olution ~20 1) prepared as described in Example 1 wa~ ultrafiltered in an ~micon DC30 hollow fiber unit u~ing Amicon BlOP10 cartridge~ with an effective filtration area of 10 sq. ~t~ per cartridge and a nim;n~l molecular retention limit of 10,000 daltons until the ~olution was c~ncentrated to a volume of 4 1.
The results in Table 2 were ~bserved upon analysis of t~e ultrafiltered material pursuant t~ ~he above-descriked methods.

71 ~`C1 ~J2 J7'1 G~ r /~

Table 1 Feed Solution Concentrate Filtrate Membrane T A280 Sp. Act.b A280a Sp. Act.~ A~80a .un No. Used ~QC~ ) (V~ 80) (VIII:C/A280) XM5~ 25 5 . 17 1 . 24 27 . 19 ~ ~ . 09 0 . 5û

2 X~50 ~5 5 . ~1 0~ 5~ 19 . ~3 C . 61 ~ . ~2

3 Xl!100~2~ 3. 71 1 . 11 16 . 91 ~ . 22 0 . 22X~lO~A. 5 5.76 ~.76 ~.31 0.73 0.3S
2~3~ 25 5. 3~ ~ . 73 73. g6 0. 77 ~ . 3û
P~ 5 5.48 0.78 33.17 0.82C 0.~3 e .

a - Absorbance at 280 n~noTr-~ters~
b - Speciic activity . indicated by units of biological activity per total protein content .
c - Although the specific activity in clear soltltion remained the same, 1596 of total VIII o C activity was lost in the precip7 tate which forl[!ed.

~1 o ~-- c~

H O O O O O O O O O r-i ~ 5!

- o r~ o O N cr~ CO ~ ~ 1~ 1` 0 0~ ~) N ~) ~ ~ I` ~r ~D X ~ U') N

N O ~ ~ a~
U~ ~ ~ t`

o ~ o a~ 1 r4 ~ ~q O ~ ~ N 11~ N ~D O
O _I N X 0~ 1~ ~ N r~4 CO
~D 1-- Sr~ r l tr) I~ ~ ~ N ~r S,~l ~ ~ ~) N

E~
~ D U') ~ r~ U~ I~ S~
O O O O O O O O O O

~ ~) I.D 1~ ~ n O O CO O
CO ~D 01) O~ O ~ I~ ~ N O O ~I
~r ~ 5 2~ ~

~ O ~ U~ O O ~

X ~ ~ ~ g s ~o g o ~

~ ~ rLI ~J trl ~ Ir~ D t~ S~ 0 - lD -~5 The ultrafiltered material was mixed with sodiu~
citrate and sodium chloride in an amount ~ufficient to attain a level of O.15 molar sodium chloride and O.01 molar sodium citrate. Next, the p~ of the constituted ma~erial was adjusted tG 6.9 by addition o 1 ~ hydrochloric acidO Then, 10 ml.
each of constituted materlal was placed in vials and freeze-dried at a pressure lower than 400 microns and a starting shelf temperature of -~0 C. to completion shelf temperature of +30 C

~xample 4 Ultrafiltratlon of ~F Solution in Hollow Fibers - Production Scale Runs Aqueous AHF solution prepared as described in Example 1 wa5 ,ultrafiltered in an ~micon DC hollow fiber unit using Amicon ~lOP10 cartridge.
The results of three runs are tabularized below.

Table 3 Production 5cale UltraPiltra~ion Runs ti ~ ,~LL d~e ~olume VIII-C ~blume VIII-C VIII-C ~Kx~ery R~n Nc). (1) (U/~)a 11~ (u/}r~)a (%) 1 99.95 4.459.80 39.~0 86 2 92.60 3.408.93 ~7.50 106 3 92.16 5.~0~6.~7 27.~0 ~7 a - U/ml = ~ ts of activity pex ~ ter.

Example 5 Protein Species Di tribution in Ultrafiltered Concen~rate The ultrafiltered concentrates from ~xample 3, ~uns 1, 2 and 39 re~pe~tively9 were subjected to cellulose acetate electrophore-~is to determine protein species distri-~u~io~.
The results are ~l ~rized in the following Table 4.

Table 4 Protein Species Distrib~ion in Ultrafil tered Concentrate VIII:C per VIII:C per R~l t~l~ll in mg. (~lQh~ll in mg.
. A1b~ 2 ~31 Y 0afi}~rir~Alhl~nin ~1 a2 ~I Y Jaaf;hrin~7~Pn 47.5 0.3 18.0 4~51.9 ~7.~ - 39.5 1.~ 23.5 4.2 2.2 29.6 3.442 38.1 --2307 ~.3 -- 35.~ -- 33.g 1.0 25.2 3.g 1.~ 34,3 3.44 36.1 0.3 ~7.7 4.0-- 3~.9 4.6 0.6 27.1 4.1 -- 33.5 3.~2 ~
r2r~pn and g32 ~ l;n have sillli~ bilities; the s~ of ~i~h i8 sh;~ as .1~.

3~

Example 6 Ultrafiltration of ~HF Solution Followed by GlyGine Precipitation Ultxafiltered AHF ~olution (8.6 1) prepared as described in Example 4 was cooled to 12 C. a~d mixed with sodium ci rate to a concentxation o~ 0.01 molar, with sodi7lm chloride to a coneentration of 0.15 molar, and with glycine to a concentration of 1.9 molar. The temperature o the mixture was lowered to 5 C. and maintained at 5 C.
throughout the treatment. ~ftex 1 hr. the mixture was centrifuged at 5 C. in a Static mode at 8500xg for 30 minutes, and a paste was separated from effluent.
The paste from glycine treatment was dissolved in final container bufer system (0.01 M sodium citrate and 0.15 M saline), an'd a portion of the solution w~ analyzed according to the aforementioned procedure~.
The remaining portion o the above solution was frozQn at -7n c. ~or 30 days, thawed, and analy2ed as above.
~ he results are ~ummaxized in the table below.
VIII:C VIIIR:WF
Sample ~280(~/ml) (~/ml) Ultrafiltered ~olution 80.6045.8 44.25 Dissolved paste after glycine treatment 47.10 33.6 3g.0 after freezing & thawing 47.10 31.7 35O0 The rozen solution xom above was thawed and prefiltered. Dextrose wa~ added to a level of 1%, and the pH of the solution was adjusted to 6.9 by addition of 1 M
hydrochloric acid. ~he sol7~tion was filtered through 0.45 ~/
0.22 ~ Pall filters to a sterile bulk tank, from which 19 ml~
each of the ~olution was plac2d in vials. The ~ontent~ of ~he ~ials were free2e-dried in a Stok@s ~reeze-dryer as de cribed above. I
Freezewdried pr~duct from one vial was constituted in inal container buffer and analyzed as described above~
~he following resulis were obtained:
8peciiic acti~ity (VIII:C ba5i5) S 0.89 ~ olubility time : 3 min., 38 ~ec.

\ ~.

VIIIR:WF (u/ml)(l:100 dilution): 22.5 VIIIR:Ag (~/ml)(1:200 dilution): 81 Clarity : ~80~

Clarity was measured by determining transmittance o f the sample at 580 nanometers. The control or standard was water, and clarity was expressed as transmittance of ~ample (580)~transmittance o wat~r ~5803x 100.

- 14 ~

Claims (62)

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

1. A process for the production of antihemophilic factor concentrate, comprising the steps of -(a) subjecting an aqueous solution of antihemophilic factor proteins to purification, (b) subjecting the purified aqueous solution of antihemophilic factor of (a) to ultra-filtration to remove water, and (c) mixing the concentrated aqueous solution of antihemophilic factor of (b) with buffer and saline.

2. The process of claim 1, which further includes the step of freeze-drying the ultrafiltered aqueous solution of step (c).

3. The process of claim 1, wherein the aqueous solution of antihemophilic factor proteins is purified in step (a) by mixing with an aluminum hydroxide adsorbent.

4. The process of claim 3, wherein the pH of the mixture is acidic.

5. The process of claim 1, wherein the ultrafiltration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off less than one million daltons.

6. The process of claim 1, wherein the ultrafiltration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off within the range of 10,000 to 300,000 daltons.

7. The process of claim 1, wherein the ultrafiltration is conducted using a hollow fibre ultrafiltration membrane.

8. The process of claim 1, wherein the ultrafiltration is conducted with a laminar flow rate at the ultrafiltration membrane wall less than Reynolds Number 2000.

9. The process of claim 1, wherein the ultrafiltration is conducted with a shear rate at the ultrafiltration membrane wall less than 2000 reverse seconds.

10. The process of claim 1, wherein the ultrafiltration is conducted using a diaphragm pump.

11. The process of claim 1, which further includes the step of mixing the ultrafiltered aqueous solution with glycine to further purify it.

12. The process of claim 11, wherein the ultrafiltration aqueous solution is mixed with glycine prior to mixing it with buffer and saline.

13. The process of claim 1, wherein the aqueous solution of antihemophilic factor proteins is obtained by solubilizing antihemophilic factor proteins from blood plasma cryoprecipitate in aqueous medium.

14. The process of claim 1, which further includes the step of adjusting the aqueous solution of step (c) to a slightly acid pH by addition of acid.

15. Antihemophilic factor concentrate produced by the process of claim 1.

16. A process for the production of antihemophilic factor concentrate, comprising the steps of -(a) subjecting an aqueous solution of antihemophilic blood plasma cryoprecipitate to purification with an aluminum hydroxide adsorhent, (b) subjecting the purified aqueous solution of anti-hemophilic factor of (a) to ultrafiltration to remove water, (c) mixing the concentrated solution of antihemophilic factor (b) with buffer and saline and adjusting to an acid pH, and (d) freeze-drying the mixture.

17. The process of claim 16, wherein the ultrafiltration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off less than one million daltons.

18. The process of claim 16, wherein the ultrafiltration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off within the range of 10,000 to 300,000 daltons.

19. The process of claim 16, wherein the ultrafiltration is conducted using a hollow fiber ultrafiltration membrane.

20. The process of claim 16, wherein the ultrafiltration is conducted with a laminar flow rate at the ultrafiltration membrane wall less than Reynolds Number 2000.

21. The process of claim 16 wherein the ultra-filtration is conducted with a shear rate at the ultrafiltration membrane wall less than 2000 reverse seconds.

22. The process of claim 16 wherein the ultra-filtration is conducted using a diaphragm pump.

23. The process of claim 16 which further includes the step of mixing the ultrafiltered aqueous solution with glycine to further purify it.

24. The process of claim 23 wherein the ultra-filtered aqueous solution is mixed with glycine prior to mixing it with buffer and saline.

25. Antihemophilic factor concentrate produced by the process of claim 16.

26. In the process for the production of anti-hemophilic factor concentrate in purified form wherein an aqueous solution of antihemophilic blood plasma cryoprecipitate is subjected to purification by mixing with an aluminum hydroxide adsorbent and by precipitating unwanted protein in the cold and wherein the aqueous solution is mixed with buffer and saline and adjusted to an acid pH, and wherein the aqueous solution is freeze-dried, the improvement which comprises concentrating the aqueous solution by removal of water prior to mixing it with buffer and saline.

27. Process according to claim 26, where the water is removed by subjecting the aqueous extract to ultrafiltration.

28. Process according to claim 27, wherein the water is removed by means of a semi-permeable membrane.

29. The process of claim 26 wherein the ultra-filtration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off less than one million daltons.

30. The process of claim 26 wherein the ultra-filtration is conducted using an ultrafiltration membrane having a nominal molecular weight cut-off within the range of 10,000 to 300,000 daltons.

31. The process of claim 26 wherein the ultra-filtration is conducted using a hollow fiber ultrafiltration membrane,

32. The process of claim 26 wherein the ultra-filtration is conducted with a laminar flow rate at the ultrafiltration membrane wall less than Reynolds Number 2000.

33. The process of claim 26, wherein the ultrafiltration is conducted with a shear rate at the ultrafiltration membrane wall less than 2000 reverse seconds.

34. The process of claim 26, wherein the ultrafiltration is conducted using a diaphragm pump.

35. The process of claim 26, which further includes the step of mixing the ultrafiltered aqueous solution with glycine

36. The process of claim 35, wherein the ultrafiltration aqueous solution is mixed with glycine prior to mixing it with buffer and saline.

37. Antihemophilic factor concentrate produced by the process of claim 26.

38. Antihemophilic factor concentrate containing Von Willebrandt factor and procoagulant factor in a ratio of about 0.5 to 1/1.

39. The product of claim 38, wherein the concentration of Von Willebrandt factor is about 15 units per milliliter or more.

40. The product of claim 38, wherein the ratio of Von Willebrandt factor to procoagulant factor is about 1/1.

41. Antihemophilic factor concentrate having a ratio of procoagulant factor to milligrams of fibrinogen of about 2.8 to 4/1.

42. Antihemophilic factor concentrate containing Von Willebrandt factor (VIIIR:WF), procoagulant factor (VIII:C) and fibrinogen (?) wherein the ratio i,s in the range of about 1/0.5 to 1 and wherein the ratio of procoagulant factor to milligrams of fibrinogen (VIII:C/mg ?) is in the range of about 2.6 to 4.0/1.

43. A process according to claim 1 including a step of recovering an antihemophilic factor concentrate contain-ing Von Willebrandt factor and procoagulant factor in a ratio of about 0.5 to 1/1.

44. A process according to claim 43 wherein said recovered concentrate has a concentration of Von Willebrandt factor of about 15 units per milliliter or more.

45. A process according to claim 43 wherein said recovered concentrate has a ratio of Von Willebrandt factor to procoagulant factor of about 1/1.

46. A process according to claim 1 including a step of recovering an antihemophilic factor concentrate having a ratio of procoagulant factor to milligrams of fibrinogen of about 2.8 to 4/1.

47. A process according to claim 1 including a step of recovering an antihemophilic factor concentrate containing Von Willebrandt factor (VIIIR:WF), procoagulant factor (VIII:C) and fibrinogen (?) wherein the VIII:C/VIIIR:WF
ratio is in the range of about 1/0.5 to 1 and wherein the ratio of procoagulant factor to milligrams of fibrinogen (VIII:C/mg ?) is in the range of about 2.6 to 4.0/1.

48. A process according to claim 16 including a step of recovering an antihemophilic factor concentrate contain-ing Von Willebrandt factor and procoacgulant factor in a ratio of about 0.5 to 1/1.

49. A process according to claim 48 wherein said recovered concentrate has a concentration of Von Willebrandt factor of about 15 units per milliliter or more.

50. A process according to claim 48 wherein said re-covered concentrate has a ratio of Von Willebrandt factor to procoagulant factor of about 1/1.

51. A process according to claim 16 including a step of recovering an antihemophilic factor concentrate having a ratio of procoagulant factor to milligrams of fibrinogen of about 2.8 to 4/1.

520 A process according to claim 16 including a step of recovering an antihemophilic factor concentrate containing Von Willebrandt factor (VIIIR:WF), procoagulant factor (VIII:C) and fibrinogen (?) wherein the VIII:C/VIIIR:WF
ratio is in the range of about 1/0.5 to 1 and wherein the ratio of procoagulant factor to milligrams of fibrinogen (VIII:C/mg ?) is in the range of about 2.6 to 4.0/1.

53. A process according to claim 26 including a step of recovering an antihemophilic factor concentrate contain-ing Von Willebrandt factor and procoagulant factor in a ratio of about 0.5 to 1/1.

54. A process according to claim 53 wherein said re-covered concentrate has a concentration of Von Willebrandt factor of about 15 units per milliliter or more.

55. A process according to claim 53 wherein said re-covered concentrate has a ratio of Von Willebrandt factor to procoagulant factor of about 1/1.

56. A process according to claim 26 including a step of recovering an antihemophilic factor concentrate having a ratio of procoagulant factor to milligrams of fibrinogen of about 2.8 to 4/1.

57. A process according to claim 26 including a step of recovering an antihemophilic factor concentrate contain-ing Von Willebrandt factor (VIIIR:WF), procoagulant factor (VIII:C) and fibrinogen (?) wherein the VIII:C/VIIIR:WF
ratio is in the range of about 1/0.5 to 1 and wherein the ratio of procoagulant factor to milligrams of fibrinogen (VIII:C/mg ?) is in the range of about 2.6 to 4.0/1.

58. Antihemophilic factor concentrate containing Von Willebrandt factor and procoagulant factor in a ratio of about 0.5 to 1/1, produced by the process of claim 43, 48 or 53.

59. Antihemophilic factor concentrate containing Von Willebrandt factor and procoagulant factor in a ratio of about 0.5 to 1/1; and having a concentration of Von Willebrandt factor of about 15 units per milliliter or more, produced by the process of claim 44, 49 or 54.

60. Antihemophilic factor concentrate containing Von Willebrandt factor and procoagulant factor in a ratio of about 1/1, produced by the process of claim 45, 50 or 55.

61. Antihemophilic factor concentrate having a ratio of procoagulant factor to milligrams of fibrinogen of about 2.8 to 4/1, produced by the process of claim 46, 51 or 56.

62. Antihemophilic factor concentrate containing Von Willebrandt factor (VIIIR:WF), procoagulant factor (VIII:C) and fibrinogen (0) wherein the VIII:C/VIIIR:WF ratio is in the range of about 1/0.5 to 1 and wherein the ratio of procoagulant factor to milligrams of fibrinogen (VIII:C/mg ?) is in the range of about 2.6 to 4.0/1, produced by the process of claim 47, 52 or 57.