The Use of Pluronic Polyols in the Precipitation of Plasma Proteins and Its Application in the Preparation of Plasma Derivatives L. A. GARCIAAND G. A. ORDONEZ From the Hyland Division of Travenol Laboratories, Inc., Costa Mesa. Caliyornia

Pluronic F-38 was used as a precipitant of plasma proteins under varying conditions of pH and polymer concentration. Results indicated that marked differences in the solubility of the plasma proteins in F-38 solutions can be applied to the separation of plasma components. The feasibility of the industrial application of this fractionation method was tested in several experiments. Conditions were established for the preparation of albumin, human plasma protein fraction ( H P P F ) , and immune serum globulin (ISG) with similar yield and purity as those prepared by the Cohn method. Current procedures for the preparation of antihemophilic A (Factor VIII) concentrate and prothrombin complex (Factors 11, V11, IX, and X) were adapted to the F-38 process by removal of the clotting factors irom the starting plasma prior to polymer precipitation. In addition, a plasma protein SOhtion free of lipoproteins, isoagghtinins, and clotting factors was developed which has proven useful as a perfusion medium in organ preservation.

widely used in the separation of plasma to largecomponents and its scale fractionation has been suggested. Recently, certain water soluble, linear, nonionic polymers have been used as precipitating agents in the preparation of antihemophilic A (Fact0.r VIII) concentrateI5.28.38 from the cryoprecipitate Of fresh plasma. The use of these polymers followed from early research applications of polyethylene glycol (PEG) (Carbowax) in the precipitation of enzymes during the isolation of chloroplasts,36 the use of a variety of high molecular weight polymers in the precipitation of protein mixtures,23 and the use of PEG in the separation of human immune Many studies which relate the solubility of a variety of proteins in high weight polymer systems have been carried Albert~sonl-~demonstrated that marked shifts in the solubilities of Proteins in polymer systems can be effected by changes in pH and ionic strength. These investigations indicated that polymer solubility conditions could be established that would be just as effective or better in.the separation of complex mixtures of proteins as those obtained by the cold ethanol method. The use of water soluble polymers of high molecular weight presents certain problems such as the difficulty in eliminating

A VAR,ETY of agents have been employed in the large scale commercial fractionation of plasma proteins for clinical use.21.3;.35 These included cold ethanol, cold cold ethyl ether, and ammonium

sulfate alone or in combination with 2ethoxy-6,9-diaminoacridine (rivanol). In ad&tion, gel adSOrption,B.30.39 ion exchange chromatography,L4.22.29.33.34 and gel filtration 10.17.19,27 have also been used to isolate proteins for eventual clinical use. preparative e~ectrophoresis5, 13.16.32 has been

Received for publication December 27, 1974, accepted March 16, 1975.

32 Transfusion Jan.-Fcb. 1976

Volume 16 Number I

cc

STOAlOd 31NOXnld H U M NOIIVNOIL3VXd

34

Transfusion Jan.-Fcb. 1976

GARCIA A N D O R D O N E Z

Materials and Methods

FIG.2. Precipitation of albumin, IgG, and IgM with Pluronic F-38 at pH 7.0 through 8.0. The percentage of the available protein in the starting material precipitated is plotted against Pluronic F-38 concentration.

weight) 80 per cent hydrophilic polyoxyethylene groups and 20 per cent of hydrophobic polyoxypropylene groups. The solubility of F-38 is greater than that of F-68 because of the lower molecular weight of its hydrophobic base. In preliminary investigations, Pluronic F-38 appeared superior to other polymer systems in the preparation of such products as AHF (Factor VIII), a Factor IX Complex, and intravenous immune serum globulin (ISG). As a result, the precipitating effect of Pluronic F-38 on plasma proteins was further investigated under different conditions of pH and polymer concentration. Based on results from this study, a fractionation scheme was designed" to prepare the same plasma derivatives that are obtained by the Cohn method.

Aliquots of plasma diluted 1:1 with normal saline were adjusted to a pH between 4.5 and 8.0 with either I N HCI or I N NaOH. Different volumes of a 60 per cent F-38 solution in saline were then added to the diluted plasma samples to bring them to a polymer concentration ranging from 2 to 35 per cent. After 30 minutes mixing a t room temperature, the precipitates were separated by centrifugation and reconstituted in 0.15M NaCl to their original volume. The resulting solutions of the precipitates and the supernatant fluids were analyzed by immunoelectrophoresis against horse anti-whole human serum. The protein concentration in the fractions was determined by either the biuret'* or Lowry's method.20 Lowry's method was run with samples showing less than 0.2 per cent protein concentration as measured by the biuret method. Quantitation of specific proteins was done by radial immunodiffusion using Hyland immunoplates. Fresh frozen plasma with a starting volume of approximately 35 liters was used in pilot fractionation experiments. Flaked pluronic F-38 was added (w/v) to the precipitating mixtures instead of the 60 per cent solution used in preliminary experiments. In most cases, the clotting factors (AHF and prothrombin complex) were removed prior to F-38 precipitation. Samples of the resulting plasma derivatives, albumin, human plasma protein fraction (HPPF), and immune serum globulin (ISG), were tested to determine whether the specifications for purity, stability, general safety, sterility, pyrogenicity, and physicochemical characteristics set by the Bureau of Biologics, FDA for therapeutic plasma products were met. In-process samples and final products were characterized as were the fractions obtained in preliminary experiments.

Results At pH 4.5 to 8.0 with a F-38 concentration greater than 35 per cent, all proteins were precipitated. As the polymer concentration is decreased, proteins begin to appear in the supernatant fluid, with the lower molecular weight proteins appearing first. Figure 1 illustrates the F-38 ranges where different plasma proteins are precipitated at various pH values. In Figures 2 and 3 are plotted the percentage of precipitation of albumin, IgG, and IgM at pH 4.5 to 8.0 versus F-38 concentrations from 2 to 35 per cent. The solubility of plasma proteins in Pluronic F38 solutions appears to be inversely proportional

Volume 16 Number I

35

FRACTIONATION WITH PLURONIC POLYOLS

to their molecular size. The higher molecular weight proteins such as IgM and a , M can be precipitated with relatively low concentrations of F-38 (4-8%), particularly a t pH range 4.5-5.0. Under these conditions, only negligible amounts of lower molecular weight proteins such as albumin and transferrin are removed. The latter proteins and other a and /3 globulins of low molecular weight require higher concentrations of F-38 for their precipitation. This is seen especially as pH increases from 4.5 to 8.0. Conversely, as pH decreases from 8.0 to 4.5, less F-38 is needed to affect the total precipitation of these proteins. The solubility of IgG differs considerably from that of albumin as the pH rises from 4.5 to 8.0. This is particularly noticeable at neutral o r higher pH, where a 12 to 14 per cent F-38 concentration is sufficient to precipitate IgG and the higher molecular weight proteins, whereas albumin and the lower molecular weight globulins remain in solution. Conversely, as pH decreases from 8.0 to 4.5, the precipitation curves of both proteins draw nearer to each other. Based on the above observations, a scheme for plasma fractionation was designed which was followed in several fractionation experiments, at laboratory and semi-industrial levels." In these experiments, developmental work was done to establish conditions for the separation of albumin, HPPF and ISG. By pretreating fresh frozen

FIG.3. Precipitation of albumin, IgG, and IgM wit Pluronic F-38 at pH 4.5 through 6.5. The percentage c the available protein in the starting material precipitated is plotted against Pluronic F-38 concentration.

SCHEME FUR I'LASMA FRACl.10Fresh Frozen Plasmn C r y o p r e c i p i t a t i o n or P r e c i p i t a t i o n w i t h 2 . 3 E: r l y c i n e , p l i 6 . 8 8 2°C

I

Precipitate P r o c e s s e d t o N1F Concentrate

FIG. 4. Schematic representation of the separation of AHF (Step A) and prothrombin complex (Step B).

I

Precipitate R e c o n s t i t u t e i n s a l i n e t o 5% proteln

Supernatant rl (l':,ctor V I I I poor plasma) D i l u t e 2 timc.; o r i E i n a l volumc w i t h saline, p!I 6 . 35; F-3M, I?oom ' T c n p c r a ~ u r c

Supern;$t i n t ( N n Ci t r n ~ e )

Discard

Cake C l o t t i n g Factors Adsorbed t o Calcium Phosphate

1

Eluted a n d p r o c e s s e d t o Prothrombin C q l e x concentrate

Supernatant R Nay bc p r o c e s s c d as i n c or D

36

GARCIA AND ORDONEZ

Transfusion Jan.-Feb. 1916

____

SCMEPIE FOR I'LASMA F M C 1 IONATION

STEP C supernntant B

Dilute to 1 % prolein w i t h 0.151 NaCl 15% F-38 pH 7.5 CI

'

7

r

=2

supe*netant

Precipitate Immunoglobulins. ' 2 macroglobulin. plasaioogen. Suspend i n saline to 1% protein pH 1 . 2 * 0 . 5 % Calcium Phosphate. ( C ~ ~ O ( O H ) ~ ( P O ~ )Rm. G ) Temp.

Albumln. transferrin. and low molecular wt. 1 . m d @ globullns. 22% F-38. pH 7.5. Room Temperature

I

Precipitate Clottlng factors adsorbed t o Calclum Phosphate

Supernalanr 1 4 % F-38 pH 1 . 0 , Room

Precipitate Suspend in saline 8% F-38. pH 4 . 5

S"per"atv"t (Some albumin) Discard

Precipitate (Igbi. IgA. some IgC. I n. ILW, Plasainogen aid t h r o d i n

supernatant 9% F-38 pH 5

Precipitate n and 8 globulins Traces of 1%:

Prec 1p1tate

supernatant Some ~lvcosrotelns

I

HPPF

supernatant 22% F-38 pH 4 . 5 . 5°C

Resuspend b i S-38

, .012 H Na Caprylate Heat 4 hours at 10%

pH 5.1.

r-------1 Precipitate (Complement Sactors)

supecnatant. pn 7 ,

I,-$

S"per"3tn"t.

Precipitate. ISG 6% ( 1 . V . ) or 1 6 . S X (1.M.)

Discard

FIG. 5 . Schematic representation of the isolation of ISG 'and albumin. The immunoglobulin containing fraction was processed to ISG for either I.V. administration (6% IVGG) or intramuscular use (Step C,). The albumin containing fraction was processed to either 5 per cent H P P F or 5 per cent albumin (Step C?). The final purification o f albumin to greater than 96 per cent was accomplished by an adaptation of the method of Porsche et a/.

precipitate Denatured n a n d B globulins

supernatant 19% F-38. pH 4 . 5 P C

I Precipitate A 1 burn1 n

plasma prior to F-38 precipitation, the clotting factors were removed and further processed for the preparation of A H F and prothrombin complex. This scheme followed the sequence given below.

Step A-Separation o f A H F Fresh frozen plasma was treated either by amino acid precipitation3' or by cryoprecipitation.2s Both precipitates were processed to render an A H F concentrate by the method of Shanbrom and FeketeZR although any of the available method^^^.^^ could have been applied as well. Step B-Separation of the Prothrombin Complex Cryoprecipitated plasma or glycine-treated plasma was diluted two times the original plasma volume with 0.15 M NaCI and precipitated with 35 per cent F-38 at pH 6. After mixing and centrifugation at room temperature, the supernatant fluid was discarded and the precipitate paste (containing the bulk of plasma proteins) was reconstituted in saline to the original volume. To the resulting suspension 0.5 per cent C a l 0 (OH),(PO,),, was added to adsorb the clotting factors.' After mixing and centrifugation, the precipitate was eluted and processed by the method of Fekete and S h a n b r ~ m .Figure ~ 4 illustrates Steps A and B.

supernatant Discard

Step C-Preparation of ISG. Albumin and HPPF Precipitation of the supernatant fluid from step B with 15 per cent F-38 at pH 7.5, yielded a precipitate fraction containing proteins with a molecular weight greater than 100,000 (immune globulins, a 2 M , plasminogen, complement components) and a supernatant fraction containing low molecular weight proteins (albumin, transferrin, haptoglobin, and other low molecular weight a and /3 globulins). The precipitate was further processed, (Step C , ) for the separation of macroglobulins and the preparation of ISG. The supernatant fraction was processed (Step C,) for the preparation of either H P P F or albumin. Step C, C,, and C, are described in Figure 5. The immunoelectrophoretic characterizations of the fractions obtained in Steps C, C , ,and C, are given in Figures 7 and 8 respectively. Step D- Preparation of a Perfusion Medium f o r Organ Preservation Supernatant fraction from Step B was processed to render an organ perfusate in some experiments. This is illustrated in Figure 8. Precipitation with 7 per cent F-38 at pH 4.5 carried down the lipoproteins, a 2 M , and IgM which were separated by centrifugation. The remaining proteinaceous material was recovered from the supernatant fraction by increasing the F-38 con-

Volume 16 Number I c

STEP

supcmut*nr B

I

151 P o l p e r pH 7 . 5

I

I

S"pcm.mnf C2

D.52 C a l d t m Phosphate pll 7 . 2

Preclplrotc Dlscord

1 4 1 Polymer pll 7 . 0

Prcclpirllre

37

FRACTIONATION WITH PLURONIC POLYOLS

I

IS(: AX (1.V.) o r 16.5% ( I . > I , I

composed of proteins with a molecular weight greater than 100,000 (macroglobulins, immunoglobulins, plasminogen, thrombin, and complement factors among others). Remaining in the supernatant fraction are albumin, haptoglobin, transferrin, and low molecular weight a and @globulins.Both the precipitate and supernatant fractions are similar in protein composition to the respective precipitate and supernatant of Cohn Fraction I I + I I I . The fact that proteins of molecular weight higher than IgG can be precipitated with relatively low F-38 concentrations at low pH range can be applied to processing the former precipitate to separate the IgG from these proteins. This step yields an ISG preparation (>90% IgG) and a precipitate similar in protein composition to Cohn Fraction 111. This precipitate as the latter fraction can be used as the starting material for the preparation of a2M, IgM, IgA, plasminogen, and thrombin. The separation of albumin from a

FIG. 6. Immunoelectrophoretic characterization of in-process fractions and final material obtained during the isolation of ISG. Immunoelectrophoresis was run against horse anti-whole human serum.

centration to 19 per cent. After mixing and centrifugation, the resulting precipitate was dissolved to 5 per cent protein in an electrolyte solution similar to that of plasma. This preparation, free of lipoproteins and coagulation factors, has been shown to be as efficient as Belzer's cryoprecipitated plasma4 in the preservation of kidneys without the inconveniences found using the latter perfusate.I8

STEP C s ~ p e r n tan a t

15% Polymer

PH 7 . 5

1 Predpitnte

pH 7 . 5

'

I 2 2 % Pnlymcr pH 4 . 5 (5%

Discussion The results from preliminary fractionation experiments with P h o n i c F-38 indicate that the different solubilities of the plasma proteins in F-38 solutions may be advantageously used for plasma fractionation. Markedly different solubilities, particularly seen at neutral pH between albumin and IgG and proteins of higher molecular weight can be used in the initial precipitation of plasma proteins with 15 per cent F-38 (w/v). This step results in the formation of a precipitate

m

I

Resuspension

5% PLASM PROTEIN

5% ALBUMIN

FIG. 7. Immunoelectrophoretic characterization of in-process fractions and final materials obtained during the isolation of albumin. lmmunoelectrophoresis was run against horse anti-whole human serum.

38

Transfusion Jan.-Feb. 1976

GARCIA A N D O R D O N E Z SCIIEElI: T O H I’1,ASMEI FRACTIIINATION ~

STEP D Superna Cant B

D i l u t e t o 1% p r o t e i n .5 g Fibr.inogen/l Mix 2 h o u r s a t room t e m p e r a t u r e . 5 g% C a l c i u m I’hoslihate ( C a 1 0 ( O H ) 2 ( P 0 4 ) 6 ) pll 7 . 2 , room t e m p e r a t u r e

p--i--

I

1’rc.c i 1, i t;i LC.

Supernatant 72 F-38 PH 4 . 3 , noom T e m p e r a t u r e

( c l o ~ t i n gf a c t o r s a d s o r h v d t o C a l c i u m P h o s p h a t e )

I) i scii rd

I

FIG. 8. Schematic representation of the preparation of a coagulation factor lipoproteins - free plasma protein perfusate.

I

Supernatant pll 4 . 5 ( a c e t a t e b u f f e r ) 1 9 2 F-38 5%

Supernatant Glycoproteins A 1 [’hal A n t i t r y p s i n

PrcripiLnre L i p u p r o t e i n - f r e e Organ P e r f u s a t e

6% P r o t e i n

each. In most cases, the clotting factors (AHF and prothrombin complex) were removed prior to F-38 precipitation. The resulting albumin-rich fraction was further processed to either 5 per cent albumin or HPPF, and the immune globulin fraction was processed to ISG for intravenous administration (6% intravenous gamma globulin [IVGG]) or intramuscular use (16.5%). Final precipitate pastes were not lyophilized but directly dissolved in pyrogen-free water, followed by pH and electrolyte adjustments, clarification, and sterile filtration. Yields of

and /3 globulins of low molecular weight, found in the 15 per cent F-38 supernatant, can be accomplished by further precipitation with higher concentrations of F-38 at pH 7.5-8.0. Under these conditions, albumin remains in solution, whereas some globulins are precipitated. An albumin-rich fraction is then precipitated from the supernatant fraction by lowering the pH to 4.5. To test the application of these findings to relatively large plasma fractionation, six experiments were performed with a starting plasma volume of approximately 35 liters

Table 1. Yield and Purity of Plasma Derivatives Obtained with Pluronic F-38

Run Number

Volume,of Plasma Run kgs

PF002 P F004 PF005 PF007 P F008 PF015

18.8 37.4 36.0 38.6 34.6 39.0

Yield g/liter

Albumin Product ~

~

5% albumin 5% albumin 5% albumin 5% albumin 5% HPPF 5% HPPF

~~

Purity Per Cent Albumin

ISG

g/liter

Per Cent Purity

95 95 96 99 84 79

6%IVGG 6% IVGG 6%lVGG 6%IVGG 6% IVGG 6%IVGG

4.26 4.15 4.1 4.34 3.76 3.03

100 100 100 100 100 100

~~

12.4 17.7 18.3 18.4 17.6 24.4

Volume 16 Number I

39

FRACTIONATION WITH PLURONIC POLYOLS Table 2. Physicochemical Characteristics and F-38Content o f Plasma Derivative Plasma Derivative

Run Number

F-38 g1100 ml

0969T003

5% albumin

PF004

1.39

Initial = 14 Nu" Heated = 12 Nu

0969T004

6% IVGG

PF004

1.34

Gelationsatisfactory

0969T005

6% IVGG

PF005

0969T007

5% albumin

PF005

0969T008

6% IVGG 5% albumin 5% HPPF

PF007

0969T011

6% IVGG

PF008

0969V002

16.5% ISG

PFO15

Lot Number

0969T009 0969TOlO

Stability

Gelationsatisfactory 1.01

PF007

0.7

PF008

0.86

1.59

Initial = 8.8 N u Heated = 8.7 Nu Gelationsatisfactory Initial = 15 N u Heated = 12 N u Initial = 20.3 N u Heated = 21 N u Gelationsatisfactory Gelationsatisfactory

Electrophoresis (Tiselius)

Ultracentrifuge

Heme Content

A l b = 95% a = 4% p = 2% y G = 100% -1.2X 10-5 cm2IVsec yG = 100% -1.6 X 10-5 cmZlVsec Alb = 96% a = 4% p = 0% -yG = 100% -0.9 X 10-5

4.5 SZOW

0.15 mgI100 ml

Alb = 89% a = 9% p = 2% yG = 100% -1.2X 10-5 cmVsec r G = 100% -1.6 X 10-5 cmZ1Vsec

6.7 SZOW 6.4 SZOW 4.6 SZOW 6.4 szow 4.5 SZOW 4.3

0.07 mg1100 ml

0.17 mgllOO ml 0.26 mg1100 ml

s20w

6.4 SZOW 6.1

szow

+Nephelometric units.

final products were comparable to those obtained by the Cohn Method. Purity and consistency of final products met Bureau of Biologics, FDA specifications for these plasma derivatives. Table 1 illustrates results of yield and purity. Table 2 presents some of the physicochemical characteristics and the residual F-38 present in these plasma derivatives. Based on this experimental work, it appears that a method of fractionation of plasma with Pluronic F-38 is feasible and that the industrial application of this process may be possible and desirable. All plasma derivatives that are currently manufactured by the Cohn method can be produced by the F-38 method with similar physicochemical characteristics, yields, and purity. However, considerable developmental research remains to be undertaken to establish the

optimal parameters for industrial scale fractionation. Factors should be examined which may result in the increase in yields of proteins with known therapeutic roles. The physical and biological properties, the stability as well as the toxicity of the F-38 processed products should continue to be studied. A workable system should be developed for the removal of the residual F-38 from these plasma derivatives. The possibility of recovering the precipitating agent from supernatant fractions should be investigated and its reuse in plasma fractionation evaluated. The distribution of HbSAgamong all fractions obtained through F-38 fractionation should be established. The outcome of these ongoing studies will undoubtedly influence the acceptability of this process in industrial fractionation of plasma.

40

GARCIA A N D O R D O N E Z

Acknowledgments The authors are grateful to Mr. Albert Moeller, Mrs. Zdenka Garciacelay, and Mr. Donald DuVall for their skillful technical assistance.

15.

References I.

2.

3.

4. 5.

6.

7.

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10.

I I.

12.

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14.

Albertsson, P.: Partition of cell particles and macromolecules in polymer two-phase systems. Adv. Protein Chem. 24:309, 1970. -: Partition of double-stranded deoxyribonucleic acid. Arch. Biochem. Biophy. Suppl. 1 :264, 1962. -: Partition studies on nucleic acids. 1. Influence of electrolytes, polymer concentration and nucleic acid conformation on the partition in the dextran-polyethylene glycol system. Acta. Biochim. Biophys. 103:1, 1965. Belzer, F. O., and S. L. Kountz: Preservation and transplantation of human cadaver kidneys: a two-year experience. Ann. Surg. 172:394, 1970. Bier, M.: Force flow electrophoresis and its biomedical applications. In Membrane Process for Industry, Birmingham, Ala., Southern Research Institute, 1966, p. 218. Brunning, P. F., and E. A. Loeliger: Prothrombal. A new concentrate of human prothrombin complex for clinical use. Br. J. Haematol. 21:377, 1971. Chun, P. W., M. Fried, and E. F. Ellis: Use of water-soluble polymers for the isolation and purification of human immunoglobulins. Anal. Biochem. 19:481, 1967. Didisheim, P., J. Loeb, C. Blatrix, and J. P. Soulier: Preparation of a human plasma fraction rich in prothrombin proconvertin, Stuart factor and PTC and a study of its activity and toxicity in rabbits and man. J. Lab. Clin. Med. 53:322, 1959. Fekete, L., and E. Shanbrom: Prothrombin complex prepared by precipitation with polyethylene glycol. US.Patent 3,560,475, 1971. Flodin, P., and J. Killander: Fractionation of human-serum proteins by gel filtration. Acta Biochim. Biophys. 63:403, 1962. Garcia. L. A,: Use of polymers in the fractionation of plasma proteins. Precontract Status Report. Contract NOI-HL-3-2970 (B) September, 1973. Gornall, A. G., C. J. Bardawill, and M. M. David: Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751, 1949. Hannig, K.: Preparative electrophoresis. In Electrophoresis, Vol. I I , New York, Academic press, 1967, p. 425. Heystek, J., H. G . J. Brummelhuis, and H. W. Kijnen: Contributions to the optimal use of human blood. Large-scale preparation of prothrombin complex. A comparison between

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Transfusion Jan.-Feb. 1976

two methods using the anion exchangers DEAEcellulose DE 52 and DEAE-Sephadex A-50. Vox Sang. 25:113, 1973. Johnson, A., J. Newman, P., and M. Karpatin: Antihemophilic factor prepared from blood plasma using polyethyl glycol. U S . Patent 3,652,530, 1972. Kerwick, R. A,, J. W. Lyttleton, E. Brewer, and E. S. Dreblow: A new type of preparative electrophoresis cell. Biochem. J. 49:253, 197 I . Killander, J.: Separation of human heme-and hemoglobulin-binding plasma proteins, ceruloplasmin and albumin by gel filtration. Acta Biochim. Biophys. 93:1, 1964. Klebanoff, G.: Personal communication. Lanchantin, G. F., and J. A. Friedmann: lsolation of human plasma prothrombin of high specific activity by gel filtration. Proc. SOC.Exp. Biol. Med. 114584, 1963. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265, 1951. Pennell, R. B.: Fractionation and isolation of purified components by precipitation methods. In The Plasma Proteins. Vol. I , New York, Academic Press Inc., 1960, p. 9. Peterson, E. A,, and E. A. Chiazze: Some experimental factors in the gradient chromatography of serum proteins. Arch. Biochem. 99:136, 1962. Polson, A,, G. M. Potgieter, J. F. Largier, G. E. F. Mears, and F. J. Joubert: The fractionation of protein mixtures by linear polymers of high molecular weight. Acta Biochim. Biophys. 82:463, 1964. -, and C. Ruiz-Bravo: Fractionation of plasma with polethylene glycol. Vox Sang. 23:107, 1972. Pool, J. G., E. J. Hershgold, and A. R. Pappenhagen: High-potency antihemophilic factor concentrate prepared from cryoglobulin precipitate. Nature 203:312, 1964. Porshe, J. D., J. B. Lesh, and M. D. Grossnickle: Recovery of serum albumin. U S . Patent 2,765,299, 1956. Ratnoff, 0. D., L. Kass, and P. D. Lang: Studies on the purification of antihemophilic factor. I I . Separation of partially purified antihemophilic factor by gel filtration ofplasma. J . Clin. Invest. 48:957, 1969. Shanbrom, E., and L. Fekete: Production of stable high-potency human antihemophilic factor using polyethylene glycol. U S . Patent 3,631,018, 1971. Schmid, K., M. B. MacNair, and A. F. Biirgi: The chromatographic separation and purification of acidic proteins on carboxylated ion exchange resins. J . Biol. Chem. 230:853, 1958.

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FRACTIONATION WITH PLURONIC POLYOLS

30. Schultze, H. E., and H. D. Matheka: Methodenunter ergeemisse der plasma protein sraktionienung. Behringwerk-Mitteilungen 28:9, 1954. 31. -: The principal methods for the fractionation of plasma proteins. In Molecular Biology of Human Proteins. Vol. I . Amsterdam-London-New York. Elsevier Publishing Company, 1966, p. 236. 32. Smolka, A. J . K., and E. F. Logan: Recovery of immunoglobulin G from Cohn Fraction I I + I I I by force flow electrophoresis. Prep. Biochem. 2:329, 1972. 33. Sober, H. A,, F. J. Gutter, M. M. WyckolT, and E. A,, Peterson: Chromatography of proteins 11. Fractionation of serum protein on anion-exchange cellulose. J. Am. Chem. SOC.78:756, 1956. 34. -, and E. A. Peterson: Chromatography of proteins on cellulose ion-exchangers. J. Am. Chem. Soc. 76:171 I , 1954. 35. Steinbuch, M.: Precipitation methods in plasma fractionation. Vox Sang. 23:92, 1972. 36. Stocking, C. R.: Precipitation of enzymes during

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41

isolation of chloroplasts in carbowax. Science 123:1032, 1956. Wagner, R. H., M. Smith, and W. D. McLester: Precipitation of Factor Vlll with aliphatic aminoacids. In The Hemophilias, International Symposium, Washington, Chapel Hill, University of North Carolina Press, 1964. Wickerhauser, M.: Large-scale preparation of Factor VIII-concentrate from frozen cryoethanol precipitate. Thromb Diath Haemorrh. Supp143:165, 1971. Willstatter, R., and H. Kraut: The aluminum gel of the formula AI(OH),, hydrate and hydrogel. Ghemische Berichte. 56:1I17, 1923. Wyandotte Chemical Corporation. In the Wonderful World of Pluronic Polyols, Wyandotte, Michigan, BASF Wyandotte Corp., 197 I .

Luis A. Garcia, Ph.D., Manager, Protein Chemistry Research, Hyland Division Travenol Laboratories, Inc., Costa Mesa, California 92626. Guido A. Ordonez, M.S., Research Associate, Kimberly-Clark Corporation, Neenah, Wisconsin 54956.