CA1054052A - Simplified method for preparation of high yield, high purity factor viii concentrate - Google Patents

Abstract

SIMPLIFIED METHOD FOR PREPARATION OF
HIGH YIELD, HIGH PURITY FACTOR VIII CONCENTRATE.

Abstract of the Disclosure A method for concentrating and purifying factor VIII by selective cold precipitation in low ionic strength solution is disclosed.

Description

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 ,j ,' ' `, .: :

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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, . -: ~ :

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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 _3_ : . , . ~ . .

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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 : .~
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~
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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 . .
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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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Claims (3)

WHAT IS CLAIMED IS:

1. The method of concentrating and purifying Factor VIII
consisting essentially of the steps 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 phrogen-free water at about 25°C to 30°C and about pH 7 for from about 30 to about 60 minutes;
removing lipids, denatured proteins and prothrobim complex from the extract solution by adsorption:
precipating fibrinogen its denatured and degraded products, without removal of significant amounts of Factor VIII from the resulting low ionic strength liquid extract by cooling the liquid to from about 1°C to about 2°C for from about one half hour to about 2 hours;
separating, stabilizing, clarifying and sterilizing the supernatant liquid containing about eight percent or more of the Factor VIII in the starting material;
lyophilizing the stabilized supernatant liquid to produce a Factor VIII concentrate which can be stored for long periods of time and which can be easily reconstituted by dissolution in distilled water or physiologic saline.

2. In a method for concentrating and purifying Factor VIII, the improvement comprising the steps of:
extracting cryoprecipitate into low ionic strength aqueous media at about normal room temperature;
thereafter precipitating fibrinogen from said low ionic strength solution substantially solely by cooling said solution to from about 1°C to about 2°C for at about one-half hour or more;
separating the resulting supernatant liquid containing at least about 80 percent of the Factor VIII in the cryoprecipitate starting material; and treating the liquid for long-term storage of a concentrate of the Factor VIII therein.

3. The method defined in Claim 2 including the additional step of purifying the Factor VIII by adding from about 3 to about 6 percent of polyol to the supernatant liquid which contains at least about 80 percent of the Factor VIII and cooling the resulting mixture to about 0 to 2°C. for from 1 to 2 hours to precipitate additional fibrinogen and its denatured and degraded products.