Note: Descriptions are shown in the official language in which they were submitted.
Specification
Several different methods have been described
for the production of antihemophilic fac-tor (AHF or Factor VIII)
for therapeutic use, e.g., selective precipita-tion, batch
absorption and elution, extraction in low ionic media and
chromatography. Chemicals most frequently used for
precipitation include alcohol, tannic acid, ammonium sulfate,
glycine, and polyethylene glycol. While purification o
Factor VIII entails the elimination of a variety of other
plasma proteins, fibrinogen is by far the most important and
troublesome of these proteins, particularly when denatured
by such processes as alcohol precipitation, freezing and
thawing. ~his denatured fibrinogen impairs filtration of AHF,
causes appreciable losses of AHF during purification steps
and decreases the solubility of the lyophilized product in
reconstituting fluid. Thus, any satisfactory method of
; ~ fying AHF requires removal of appreciable quantities of
fibrinogen. The selective precipitation techniques described
above are designed for this purpose but all have the
disadvantages of either further denaturing fibrinogen and AHF
or producin~, undesirable losses of AHF.
Methods utilizing simple cold precipitation
without chemicals (cryoprecipita~ion) are limlted to small scale
production usually in blood banks, and result in high I ibrinogen
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1 blood levels when used therapeutically, a feature considered
2 undesirable by so~e experts in the field.
3 Procedures which involve the extraction of Factor VIII from
4 cryoprecipitate in low ionic strength buffers, while decreasing
~ibrinogen content of the final product somewhat, still hc~ve
G an u~desirably high protein content, require special equipmen~
q and procedures Eor centrifugation and are limited -in the totaL
8 amo-mt o~ AHF which may be extracted from the cryoprecipitate ~ ;
9 without împairing purification.
Other problems commonly associated with large scale
ll manufacture o~ A~ are the contamination by pyrogenic substanc~s
12 and viruses which cause hepatitis ~hepatitis associated antigen, `
13 HAA) of the final product. With chemical precipitants these
14 undesirable contaminants may actually be enhanced.
The method herein described virtually eliminates these
16 problems, lacks the disadvantages associated with chemical
17 precipitants and relies on the simple procedure of selective
18 cold precipitation of fibrinogen, its denatured and degraded
19 products. ~Reference to removal of fibrinogen hereinafter
includes removal of-fibrinogen and its dana~ured and degraded
21 products.)
22 The selecti~e precipitation of fibrinogen without associated
23 loss of Factor VIII has not been previously accomplished as a
24 practical method for large scale matlufacture of a purified
AHF concentrate. In fact Wickerhauser (1), emphasizes the
26 importance of limiting the time and temperature in extracting
27 AHF. "Somewhat higher yields of A~ were obtained by prolonged
28 Tris extraction at 30C beyond 60 minutes, but the extract
~9 was increasingly contaminated with aggregated fibrinogen which
made the final concentrate poorly filterable. Conversely,
31
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1 lower and variable A~ yields were obtained at shorter extraction,
or at lower temperature." The procedure herein described
3 elimlna~es all of these problems because any excessive fibrinogen
contaminat-ion is removed during the cooling, while nohe of the
5 ~IF is lo.st.
6 t~hile the effect of cooling has been previously noted by
7 ~lershgoLd et al (2) they required 18 to 30 hours at 4C and
8 failed to obtain or suggest the accelerated and selective
9 precipitation of ibrinogen and isohemagglutinins at the lower
temperature oE 0-2C. (In fact, in later work Hershgold et
11 al totally eliminate a cooling step in an attempt to produce
12 very highly purified AHF (3).) The authors (2) admit that they
1~ were not able to produce a therapèutic concentrate which could
14 be consistently sterile filtered and they had to resort to
further steps of alcohol or glycine precipitation. The striking
16 improvements of the present invention were an unexpected ~``
17 surprise in view of the discouraging reports of Hershgold and
18 others.
19 James and Wickerhauser (5) comment "Furthermore3 since the
method has to be carried out at room temperature to avoid precipi-
21 tation of fibrinogen," but they failed to develop any procedure
22 to produce a Factor VIII concentrate using the prxnciples of
23 this invention. Their final product was also much lower in
24 yield and purity compared to that described in this invention.
Further processing with PEG to remove ~ibrinogen and increase
26 purity resulted in even more striking losses of Factor VIII. ~1,6)
2~ Thus, it appears that other workers have long assumed or
28 concluded from their experience that there were no particular
29 improvements in results to be expected from the use of the
only slightly lower temperatures ilsed in the precipitation
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and removal of fibrinogen, after only a very short precipitating
time. The unexpec-ted and vastly improved results found and
reported here are contrary to the teachings and suggestions
of the prior art, and were arrived at .in initial stages by the
inventor more by coincidence than by design.
Thus, in accordance with the present teaching,
an improvement is provided in the method of concentrating and
purifying Factor VIII. The im2rovement comprises extracting
cryoprecipitate into low ionic strength aqueous media at
o about normal room temperature, precipitating fibrinogen
from the low ionic strength solution substantially solely by
cooling the solution to from about 1C to about 2C for
about one-half hour or longer, separating the resulting ~ :
supernatant liquid containing at least about 80 percent of
the Factor VIII in the crvoprecipitate starting material
and treating the liquid for long-term storage of a concentrate
of the Factor VIII therein.
In accordance with a preferred embodiment there is
provided a method of concentrating and purifying Factor VIII
o which consists essentially of collecting cryoprecipitate
from about 100 or more liters of frozen plasma, extracting
the cryoprecipitate thus collected in from about 2 to about
3 volumes of pyrogen-free water at about 25C to 30C and
: at about pH 7 for about 30 to 60 minutes, removing lipids,
denatured proteins and prothrobim complex from the extract ~ :
solution by adsorption, precipitating fibrinogen its denatured
and degraded product without removal of significant amounts
of Factor VIII from the resulting low ionic strength liquid ;~
extract by cooling the liquid to from about 1C to about 2C ;
.:0 for from about one-half hour to about 2 hours, separating,
stabilizing, clarifying and sterilizing the supernata.nt liquid : ;
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containing about ei~hty percent or more of Factor VIII in
the starting material, lyophilizing the stabilized supernatant
liquid to produce a Factor VIII concentrate which may be stored
for long periods of time and which can be easily reconstituted
by dissolution in distilled water or physiologic saline.
This invention is a specific method for the large-
scale manufacture of a Factor VIII concentrate. Among the
more unique features of the invention is the selective cold
precipitation of excessive amounts of fibrinogen, its denatured ~ ;
.0 forms and degradation products in low ionic strength solution,
without added chemicals, and without undesirable loss of AHF
activi.ty. An additionally outstanding feature is the surprising-
ly high yield of Factor VIII, approximately 25-40% of the
theoretical plasma. In addition, and in spite of the high
AHF recovery, the protein content has been reduced 50-75%
compared to other products. This is illustrated in Table I.
The need for a high yield, high purity freeze-dried AHF
concentrate has received international recognition and concern .
(7,8), and it is generally agreed that commercial concentrates
0 usually yield less than 20% of the theoretical AHF present in
`. plasma (7,8,9). .: ~
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Example Method
Frozen plasma, e.y., 100 to 3000 liters, are
thawed at from about -5C to about +2C and collected in an
appropria-~e tank or vessel. Greater volumes may be handled
but operations become diEEicul~. The colcl insoluble fraction
(cryoprecipitate) is collec-ted, preferably in con-tinuous
Elow centrifuges (Sharpless or similar centrifuges), a-t less
than 3C. Other collection methods may be used but are less
efficient. The cryoprecipitate is weighed and then mixed
with a small amount of distilled, pyrogen-free water, preferably
in a Waring-type blender for a few seconds, to produce a
slurry or emulsion. The slurry is then extracted in from
2 to about 3 volumes of distilled pyrogen-free water at about
pH 7.0 for from about 30 to about 60 minutes after warming
to 20-30C. Aluminum hydroxide gel is then added to the amount
of from 10 to 30 ml. per liter and allowed to adsorb for 15
minutes. Tricalcium phosphate, 0.5-2.0~, by weight, may be
added for further purification and the amount of aluminum
hydroxide reduced. This step aids in the removal of lipids,
20 denatured proteins and prothrombin complex. The entire -
procedure is carried out in a jacketed reaction vessel with
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continuous stirring, taking care to avoid foaming. The
; contents of the vessel is then cooled to internal temperature
. , .
; of from about 1C to about 2C for from about 1/2 to 2 hours.
The heavy precipitate which forms is removed by continuous-flow -~
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centrifugation (e.g. Sharpless). The supernatant is stabilized
with 0.02 molar trisodium citrate and 0.1 molar glycine at ~-
temperature of 20-25 and the pH is adjusted to 7.0 with citric
acid. The solution is then clarified and sterilized by passing
the liquid through 293 mm Millipore~ membrane filters (or
cartridge equivalents) having, typically, 1.2, 0.65, 0.~5, or 0.3 ~ `
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micron diameter pores. The reswlting sterile solution, containing
from about 25 percent to 40 percent of the Factor VIII in the
original plasma starting material is lyophilized in the normal
manner for storage.
Varia-tions of manufacturing technique, particularly
employing reErozen cryoprecipi-tate or variants of Cohn Fraction
I may be employed, although in such situations the yield of
Factor ~III will be lessened and is dependent on its concentration
in the starting material. Extraction of AHF may be carried out
in other low ionic streng-th buffers such as Tris buffer. Cooling
time may also be varied, increased or performed repetitively
with some further aggregation of Factor VIII without departing
from the invention. Likewise, extraction time can be increased
to up to 24 hours within the procedure described.
The resultant product can be stored at +5C for
long periods of time, at least one year and reconstituted in
i distilled water or physiologic saline. Because of its
relatively low fibrinogen content and higher albumin content,
it goes into solution very quickly. Since the entire processing `
time is very short compared to other methods of manufacture
from the time that the bags of plasma are opened, bacterial
growth is limited. The fact that the extraction is carried out
` in distilled water decreases the amount of fibrinogen and
gamma globulins which go into solution. By cooling at 2C,
selective precipitation of excessive fibrinogen and its de-
natured and degraded products and isohemagglutinins (macro-
globulins) occurs without measurable loss of AHF. In addition,
the heavy flocculent precipitate of fibrinogen probably entraps
any pyrogenic material present and reduces amounts of hepatitis
~30 associated antigen (HAA or hepatitis virus). This results in
a product purified thirty to sixty times, over plasma, and very
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low in fibrinogen and "saline" isohemagglutinins. If desired,
this produc-t can be Eurther purified and concentra-ted by known
procedures or new procedures. In an exemplary new procedure,
Factor VIII preparation of extremely high purity may be
manufac-tured by an additional s-tep which includes the addi-tion
of 3-6~ polyol (PEG or Pluronic~ ~a/) and then cooling again
for 1-2 hours at 0-2C. See entry No. (7), Table I. One great
advantage of this invention is that the extremely pure AHF
can be produced in less than eight hours, as compared with
0 two to three times that long experienced with prior procedures.
The example given before is the optimum
procedure presently known for carrying out the invention. It
will be apparent, however, that the invention may be practiced
through the application of conventional processing techniques
and materials to the principle of the invention. For example,
while it is generally not economically practicable to start
- with less than about 100 liters of plasma, or cryoprecipitate
from this amount of plasma, there is obviously no criticality ~;
;~ to either the upper or lower volume values given in the example
0 and variations by fifty percent or so in these values would
not effect the invention. The procedures in the optimum example
are carried out using well-known and readily available equipment,
such as the Sharpless centrifuge, the Waring blender, etc., but -~
all will recognize that these steps per se, in isolation from
the inventive principle, and the equipment involved are not
critical and great variation can be made within the invention
within the discretion of the operator, depending upon
available manpower, equipment, etc. Adsorption of aluminum
hydroxide is, per se, a well-known step and not critical to
0 the inventive concept. Considerable discretion may be exercised
in carrying out this
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1 step through the substi~ution of other ~dsorbents, etc., or the
2 accomplishmen~ of the same objective through an equivalent step
~ or simply omitting it. Once the supernatan~ liquid, containing
4 the Factor VIII in high recovery, is obta-Lned, it is treated in
the conventional.manner or storage and reconstitution; e.g., it
6 ls clarifled and ste~rillzed, through stnndard micropore ~iltration,
7 lyoph;llzed ko concentra~e the Factor VIII into a small, easily
8 storab~e volume, and reconstituted using conventional liquids,
9 e.g. pyrogen-free water or physiologic saline.
The invention is limited by the claims set ~orth hereinater,
11 and not by the specific details o~ the exemplary procedure set
12 ~ forth the speciEication as the best mode.
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References Cit:ed in the Spe_icat:ion
1 . Wickerhauser , P~.: I.arge scale fractionation oE Factor
VIII - Concenl:rate from cryoethenol precipitate.
Thromb Diath ~laemorrh. 43:165, 1971
1~
2. ~Icrshgold, E~3., Pool, J.G., and Papperhag~n, A.R.
The potent antihemophilic concentrate derived from a cold
insoluble fraction of human plasma; Characterization and
further data on preparation and clinical trial. J. Lab.
Clin. Med. 67: 23 196~
~ `
3. Hershgold, E.J., Davison, A.M., & Janzen, M.E. Isola~:ion
and some chemical properties of human Factor VIII
(antihemophilic actor). J. Lab. Clin. Med., 77: 185,1971
4. Shan~rom, E. & Fekete, L. Production of stabLe high potency
human~AHF using polyethylena glyeol and glycine to fractionate
a eryopreeipitate of AHF eoncentrate. U.S: Patent No~
3?631 018, Dec. 28, 1971
5. James, H.L. and Wickerhauser, M.: Development of large ~;
seale fraetionation methods. Vox Sang. 23:402, 1972
-~` 6. Sgouris, J;T. and IJickerhauser, M.: Use oE frozen
eryoprecipitate~for the preparation of clinical Faetor
VIII eoncentr~te. Transfusion 13:399, 1973
. .
:
7. Recent Advances in Hemophilia. Arm. N.Y. Acad._Sci.
240, 1 4~6, 1975. ~
8. Pool, J.G. Recent chapters in the Factor VIII saga: perils ~ -
of a protein. West. J. of Med. 122:405, 1975
9. Fekete, L~F. and Holst, S.L. Stabilization of AHl~ using
- Heparin. U.S._ Patent No. 3 `803,115, April 9, 1974 ~;
~,. 10. Alpha-hydro-omega-hydroxy-poly(oxyethylene)polytoxypropylene)
poly(oxyethyene) block copolymer, BASF-Wyandatte Co;~: The
Wonder~ul World of Pluronic Polyols, 1971. PEG is the
abbreviation Eor polyethylene glycol.
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