Note: Descriptions are shown in the official language in which they were submitted.
/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~
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~'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
-- 5 --
,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.
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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 /~
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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.
- 13 -
\ ~.
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 ~
