the use of pluronic polyols in the precipitation of plasma proteins and its application in the...

The Use of Pluronic Polyols in the Precipitation of Plasma Proteins and Its Application in the

Preparation of Plasma Derivatives

L. A. GARCIA AND 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

widely used in the separation of plasma components and its to large-

solubility of the plasma proteins in F-38 solutions can be scale fractionation has been suggested. applied to the separation of plasma components. The fea- Recently, certain water soluble, linear, sibility of the industrial application of this fractionation method was tested in several experiments. Conditions nonionic polymers have been used as precipi- were established for the preparation of albumin, human tating agents in the preparation of antihemo- plasma protein fraction (HPPF) , and immune serum philic A (Fact0.r VIII) concentrateI5.28.38 globulin (ISG) with similar yield and purity as those prepared by the Cohn method. Current procedures for the from the cryoprecipitate Of fresh preparation of antihemophilic A (Factor VIII) con- plasma. The use of these polymers followed centrate and prothrombin complex (Factors 11, V11, IX, from early research applications of poly-

ethylene glycol (PEG) (Carbowax) in the 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 SO- precipitation of enzymes during the isolation htion free of lipoproteins, isoagghtinins, and clotting of chloroplasts,36 the use of a variety of high factors was developed which has proven useful as a perfusion medium in organ preservation. molecular weight polymers in the

precipitation of protein mixtures,23 and the use of PEG in the separation of human im-

the solubility of a variety of proteins in high

cold ethyl ether, and ammonium been carried Albert~sonl-~ dem- onstrated 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

problems such as the difficulty in eliminating

A VAR,ETY of agents have

fractionation of plasma proteins for clinical been employed in the large scale commercial mune Many studies which relate

use.21.3;.35 These included cold ethanol, cold weight polymer systems have

sulfate alone or in combination with 2- ethoxy-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, ac- of high molecular weight presents certain cepted March 16, 1975.

32 Transfusion Volume 16 Jan.-Fcb. 1976 Number I

cc STOAlOd 31NOXnld HUM NOIIVNOIL3VXd

Transfusion Jan.-Fcb. 1976 34 GARCIA A N D O R D O N E Z

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 polyox- yethylene groups and 20 per cent of hydrophobic polyoxypropylene groups. The solubility of F-38 is greater than that of F-68 be- cause 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.

Materials and Methods Aliquots of plasma diluted 1:1 with normal

saline were adjusted to a pH between 4.5 and 8.0 with either IN HCI or IN 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 immuno- plates.

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 ap- pearing 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 F- 38 solutions appears to be inversely proportional

Volume 16 Number I 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 a t 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, a t 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

35

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 F U R I'LASMA FRACl.10-

Fresh Frozen Plasmn Cryoprecipi tat ion 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 , pl i 6 . 8 8

2°C

I

p o o r p lasma) P r e c i p i t a t e Supernatant rl (l':,ctor V I I I Processed t o N1F

D i l u t e 2 timc.; or iEinal Concentrate FIG. 4. Schematic rep- volumc with saline, p!I 6 .

resentation of the sepa- 35; F-3M, I?oom ' T c n p c r a ~ u r c

ration of AHF (Step A) and prothrombin complex

Supern;$ t i n t

I (Step B). P r e c i p i t a t e

Reconst i tute i n s a l i n e t o 5% proteln (Nn Ci t r n ~ e ) Discard

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

Calcium Phosphate

1 Eluted and processed t o Prothrombin C q l e x concentrate

Supernatant R Nay bc processcd 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

=2 CI

Precipitate 7' supe*netant

Immunoglobulins. ' 2 macroglobulin. Albumln. transferrin. and plasaioogen. Suspend i n saline to low molecular wt. 1 . m d @ 1% protein pH 1 . 2 * 0 . 5 % Calcium globullns. 22% F-38. Phosphate. ( C ~ ~ O ( O H ) ~ ( P O ~ ) G ) Rm. Temp. pH 7.5. Room Temperature

r I

Precipitate Supernalanr Clottlng factors 1 4 % F-38 adsorbed to pH 1 . 0 , Room Calclum Phosphate

Precipitate S"per"atv"t Suspend in saline (Some albumin) 8% F-38. pH 4 . 5 Discard

Precipitate supernatant (Igbi. IgA. some IgC. 9% F-38 I n. ILW, Plasainogen pH 5 aid throdin

Precipitate supernatant n and 8 globulins 22% F-38 Traces of 1%: pH 4 . 5 . 5°C

Prec 1p1tate supernatant I Some ~lvcosrotelns

Resuspend HPPF b i S-38

.012 H Na Caprylate pH 5.1. Heat 4 hours at 10%

r-------1 , Precipitate supecnatant. pn 7 , precipitate supernatant (Complement Sactors) Denatured n a n d B 19% F-38. pH 4 . 5

globulins P C

I,-$ S"per"3tn"t. I Precipitate. Discard Precipitate ISG A 1 burn1 n 6% ( 1 . V . ) or 16.SX (1.M.)

plasma prior to F-38 precipitation, the clotting factors were removed and further processed for the preparation of AHF 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 cryoprecipi- tation.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

FIG. 5 . Schematic rep- resentation 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/.

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, a2M, 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 HPPF 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 for 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 FRACTIONATION WITH PLURONIC POLYOLS 37

STEP c supcmut*nr B

151 P o l p e r I pH 7 . 5 I

I S"pcm.mnf

C2

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

1 4 1 Polymer pll 7 . 0

P r e c l p l r o t c Dlscord

Prcclpirllre I

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

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

Discussion The results from preliminary fraction-

ation experiments with P h o n i c F-38 indi- cate that the different solubilities of the plasma proteins in F-38 solutions may be ad- vantageously 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

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

STEP C s ~ p e r n a tan t m

15% Polymer PH 7 . 5

1 pH 7 . 5 '

P r e d p i t n t e I

22% Pnlymcr pH 4 . 5 (5%

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 GARCIA A N D ORDONEZ Transfusion Jan.-Feb. 1976

SCIIEElI: TOH 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 F i b r . i n o g e n / l

Mix 2 h o u r s a t room t e m p e r a t u r e

pll 7 . 2 , room t e m p e r a t u r e . 5 g% Calc ium I’hoslihate (Ca10(OH)2(P04)6)

FIG. 8. Schematic rep- resentation of the prepa-

72 F-38 ration of a coagulation

I S u p e r n a t a n t

PH 4 . 3 , noom Temperature factor lipoproteins - free

1’rc.c i p--i-- 1, i t;i LC.

( c l o ~ t i n g f a c t o r s adsorhvd t o Calc ium P h o s p h a t e ) I) i scii rd

plasma protein perfusate. I I

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

P r c r i p i L n r e 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

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

S u p e r n a t a n t G l y c o p r o t e i n s A 1 [’hal A n t i t r y p s 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

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

Volume,of Purity Plasma Run Yield Per Cent Per Cent

Run Number kgs Albumin Product g/liter Albumin I SG g/liter Purity ~ ~ ~~ ~~

PF002 18.8 5% albumin 12.4 95 6%IVGG 4.26 100 P F 004 37.4 5% albumin 17.7 95 6% IVGG 4.15 100 PF005 36.0 5% albumin 18.3 96 6%lVGG 4.1 100 PF007 38.6 5% albumin 18.4 99 6%IVGG 4.34 100 P F 008 34.6 5% HPPF 17.6 84 6% IVGG 3.76 100 PF015 39.0 5% HPPF 24.4 79 6%IVGG 3.03 100

Volume 16 Number I

FRACTIONATION WITH PLURONIC POLYOLS 39

Table 2. Physicochemical Characteristics and F-38 Content of Plasma Derivative

Plasma Run F-38 Electrophoresis Ultra- Heme Lot Number Derivative Number g1100 ml Stability (Tiselius) centrifuge Content

0969T003

0969T004

0969T005

0969T007

0969T008

0969T009

0969TOlO

0969T011

0969V002

5% albumin

6% IVGG

6% IVGG

5% albumin

6% IVGG

5% albumin

5% HPPF

6% IVGG

16.5% I SG

P F 004

P F 004

PF005

PF005

PF007

P F 007

P F 008

PF008

PFO15

1.39

1.34

1.01

0.7

0.86

1.59

Initial = 14 Nu" Heated = 12 Nu

Gelation- satisfactory


Initial = 8.8 Nu Heated = 8.7 Nu


Initial = 15 Nu Heated = 12 Nu Initial = 20.3 Nu Heated = 21 Nu



Alb = 95% a = 4% p = 2%

yG = 100% -1.2X 10-5 cm2IVsec yG = 100%

cmZlVsec Alb = 96% a = 4% p = 0%

-yG = 100% -0.9 X 10-5

-1.6 X 10-5

Alb = 89% a = 9% p = 2%

yG = 100% -1.2X 10-5 cmVsec r G = 100%

cmZ1Vsec -1.6 X 10-5

4.5 SZOW

SZOW

SZOW

6.7

6.4

4.6 SZOW

6.4 szow

4.5 SZOW

4.3 s20w

6.4 SZOW

6.1 szow

0.15 mgI100 ml

0.07 mg1100 ml

-

0.17 mgllOO ml

0.26 mg1100 ml

+Nephelometric units.

final products were comparable to those obtained by the Cohn Method. Purity and con- sistency of final products met Bureau of Bio- logics, 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 possi- bility of recovering the precipitating agent from supernatant fractions should be investigated and its reuse in plasma fractionation evaluated. The distribution of HbSAg among 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 ORDONEZ Transfusion Jan.-Feb. 1976

Acknowledgments The authors are grateful to Mr. Albert Moeller, Mrs.

Zdenka Garciacelay, and Mr. Donald DuVall for their skillful technical assistance.

References I . Albertsson, P.: Partition of cell particles and

macromolecules in polymer two-phase systems. Adv. Protein Chem. 24:309, 1970.

2. -: Partition of double-stranded deoxyribo- nucleic acid. Arch. Biochem. Biophy. Suppl. 1 :264, 1962.

3. -: 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.

5. Bier, M.: Force flow electrophoresis and its biomedical applications. In Membrane Process for Industry, Birmingham, Ala., Southern Re- search Institute, 1966, p. 218.

4.

6 .

7.

8.

9.

10.

I I .

12.

13.

14.

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. Sou- lier: 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 Elec- trophoresis, 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

15.

16.

17.

18. 19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

two methods using the anion exchangers DEAE- cellulose 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. US. 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 he- moglobulin-binding plasma proteins, cerulo- plasmin 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. Pap- penhagen: 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.

Volume 16 Number I

FRACTIONATION WITH PLURONIC POLYOLS 41

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 . Am- sterdam-London-New York. Elsevier Pub- lishing Company, 1966, p. 236.

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.

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.

Steinbuch, M.: Precipitation methods in plasma fractionation. Vox Sang. 23:92, 1972.

Stocking, C. R.: Precipitation of enzymes during

32.

33.

35.

36.

isolation of chloroplasts in carbowax. Science 123:1032, 1956.

Wagner, R. H., M. Smith, and W. D. McLester: Precipitation of Factor Vll l with aliphatic ami- noacids. In The Hemophilias, International Symposium, Washington, Chapel Hill, Uni- versity of North Carolina Press, 1964.

38. Wickerhauser, M.: Large-scale preparation of Factor VIII-concentrate from frozen cryo- ethanol 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 Won- derful World of Pluronic Polyols, Wyandotte, Michigan, BASF Wyandotte Corp., 197 I .

37.

39.

40.

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, Kim- berly-Clark Corporation, Neenah, Wisconsin 54956.

the use of pluronic polyols in the precipitation of plasma proteins and its application in the...

Documents