FI63674C - PROCEDURE FOR INSULATION AND IMMUNOLOGICAL ACTIVATION OF ACTIVE POLYRIBOSYLRIBITOL PHOSPHATE (PRP) - Google Patents

Description

E3F * 1 M (11) ^ UUL UTUSJ U LKAISU ί-7, ηΔ LJ '' utlAqgninosskrift 000/4 (51) Kv.ik.3 / ι «.α3 A 61 K 39/102, C 12 P 19/04 FINLAND —FINLAND (21) P »Mntclh« kemu »-PM« i * M * ekiiln * 783269 (22) Hakemiiptlvl —AMBknint ^ tg 26.10.78 (23) AlkupUvft — GUtlghtadti 26.10.78 (41) Tullut {ulkMal - Bllvlt off «NTH | 29. Ol. 79 _ ^,. (44) NihUvikalpanon | t kuL) ulk ^ * un pvm. -, "

Patent and registration authorities Amekw utiagd och uti ^ kriftwi puuic «rad 29 · 0 ^. 83 (32) (33) (31) Requested Muoikeu * -B * jtrd prtortm 28.10.77 22.12.77 USA (US) 81 + 6166, 816188 (71) American Cyanamid Company, Wayne, New Jersey, USA (US) ( 72) Joseph S.-C. Kuo, Orangeburg, New York, USA (7l) Oy Kolster Ab.

(51) Method for isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP) - Förfarande för isolering och rening av immunologiskt aktivt polyribosylribitolfosfat (PRP)

The invention relates to a process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), a Haemophilus influenzae type b capsular polysaccharide. Polyribosyl rib toliphosphate can be used for immunization against Haemophilus influenzae type b infections such as meningitis. PRP prepared according to the invention can be used in combination in vaccines together with B. pertussis antigen. PRP is also likely to be effective when used in combination with other, non-pathogenic bacterial strains, such as E. coli, B. subtilis, S. aureus, B. pumilis, and L. plantarum, to elicit an antibody response in warm-blooded animals.

Purified polyribosylribitol phosphate (PRP) from Haemophilus influenzae type b is currently being studied as a protective immunogen. However, no active antigen response has been obtained in young animals and children. Ethanol and Cetavlon (hexadecyltrimethylammonium bromide) were used in the previously known purification method. Impurities, endotoxins and pyrogens were removed with cold phenol or chloroform and tert-butanol, which could result in a change in the antigenic nature of this polysaccharide.

2 63674

A new method for isolating and purifying immunologically active PRP from Haemophilus influenzae type b has now been developed.

The present invention has clear advantages over previously known methods in that all impurities (nucleic acids, proteins and endotoxins) are removed very well by treatment with hydroxylapatite. The polysaccharide obtained by the method described here has a higher molecular weight than the polysaccharides obtained by known methods.

In addition, it is of great importance that the PRP prepared by this new method is highly immunogenic in warm-blooded animals in contrast to the PRP prepared by the previously known methods.

Removal of impurities (proteins, nucleic acids and endotoxins) from the polysaccharide PRP is accomplished by treating the partially purified polysaccharide with a phosphate-containing adsorbent that does not absorb the polysaccharide under the conditions reported.

(I) Isolation and purification of polyribosyl ribitol phosphate, Haemophilus influenzae type b capsular polysaccharide Organisms, culture media and cultures Two Haemophilus influenzae type b strains were used. The rab strain was obtained from Grace Leidy Hospital, Babies Hospital, Colombia University, New York City, New York. The CK strain was isolated from a patient at Waterbury Hospital in Waterbury, Conn. They underwent several passages in mice to ensure virulence. The organisms were isolated from the brain tissue of killed mice and further cultured with either 3.7% brain-heart infusion medium (BHI) (Difco Lab., Detroit, Mich.) Or 5% BHI agar supplemented with 0.01% nicotinamide. adenine dinucleotide (NAD) (PL Biochemicals, Milwaukee, Wis.) and 1% (v / v) defibrinated horse blood (Animal Blood Center, Syracuse, NY) and then dispensed in 1 ml ampoules, lyophilized and stored - 70 ° C.

The basic medium used for culturing the organisms was 3.7% BHI. The stock medium was supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per liter. For 50 ml of inoculum, 1% (v / v) defibrinated horse blood was added. Prior to use, fresh complement is prepared and filtered through a 0.45 μm Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.). When culturing the organism in a 14-liter fermentor,

II

An additional 0.5% glucose is added to 3,63674.

To prepare an inoculum from the organisms, 1 ml of a frozen stock culture is thawed and 50 of any enriched BHI medium supplemented with difibrinated horse blood and NAD are added. The culture is grown for eight hours at 37 ° C in a gyro shaker at 150 rpm. The organisms are then added in 1% inocula to 500 ml tn portions of enriched BHI culture medium in 2 liter flasks, and the cultures are incubated at 37 ° C with gentle shaking in a gyro shaker for eight hours (to isolate PRP) or 14 hours (inocula).

Fermentation of each batch in a 14-liter fermentor is initiated by aseptically introducing 700 ml of inoculum into 7 liters of enriched BHI culture medium supplemented with hemin instead of de-fibrinated horse blood. The culture temperature is kept constant at 37-1 ° C. The tank is agitated at 150 rpm and an air flow of 0.25 liters of air / min / 1 liter of vilhely mixture is introduced. During culture, 0.001% silicone antifoam (FD-82, Hodag Chemical Corp.) is added as needed. The culture is continued to g late logarithmic growth phase (8-10 x 10 viable cells / ml), usually for about eight hours, after which the culture is stopped by adding 0.4% formaldehyde and taking the cultures overnight at 4 ° C.

Isolation of polyribosyl ribitol phosphate (PRP)

The culture medium prepared as described above is centrifuged at 27,000 g for 30 minutes at 4 ° C. The cell-free supernatant is recovered and treated as follows:

Step 1, ethanol precipitation

Sodium acetate (final concentration 4%) is added to the culture supernatant. The pH of the solution is adjusted to 6.0-6.2 and 44 liters of 3A-ethanol are slowly added with vigorous stirring at 4 ° C. The pH of the mixture is adjusted to 6.8 with glacial acetic acid and the mixture is then stored for 12 hours at 3 ° C. The precipitate formed is recovered by decantation and centrifugation to give crude PRP.

4,63674

Step 2, Cetavlo (hexadecyltrimethylammonium bromide) treatment

The precipitate from step 1 is dissolved in pyrogen-free distilled water and centrifuged to separate the insoluble matter. The light brown solution is then added slowly with stirring to 1 to 0 ml of 10% aqueous Cetavlon solution (final concentration 0.5%). The mixture is stirred for one hour, then centrifuged. The precipitate consisting of the nucleic acids and the PRP-Cetavlon complex is mixed with 2 liters of 0.3 M sodium chloride solution. The cloudy solution is centrifuged to remove insoluble material such as the nucleic acid Cetavlon.

The supernatant is diluted with an equal volume of water to precipitate the PRP-Cetavlon salt. The mixture is stirred for one hour. the precipitate is collected by centrifugation and then dissolved in 2 liters of 0.3 M sodium chloride solution.

Step 3. Ethanol precipitation

Cetavlon and nucleic acid and protein impurities are further removed by ethanol precipitation (at least twice). PRP is precipitated according to step 1, while Cetavlon is dissolved in an alcoholic solution. The PRP is recovered by centrifugation, dissolved in 2 liters of pyrogen-free distilled water and then reprecipitated as before. The final PRP precipitate is dissolved in 20 mM sodium phosphate, pH 6.8.

Step 4, Treatment of hydroxylapatite

Impurities in partially purified PRP preparations (such as nucleic acids, proteins, and endotoxins are removed by selective adsorption to a calcium phosphate-containing adsorbent such as hydroxyl lipatite / 3Ca (PO2) ^ * Ca (OH)).

The invention is based on the finding that the polysaccharide PRP is not adsorbed to a calcium phosphate adsorbent containing a phosphate buffer (molar) (pH 6.7 to 6.9) or a phosphate buffer (50 mmol) having a pH of about 5.8; impurities (such as nucleic acids, proteins, and endotoxins) are instead adsorbed under these conditions. The process according to the invention can be carried out batchwise or as a column run. In a batch process, hydroxylapatite is added to a partially purified PRP preparation (in 20 mM phosphate buffer, pH 6.9).

63674

The mixture is mixed thoroughly and centrifuged to remove unwanted solids (adsorbent and compounds adsorbed by the adsorbent). The supernatant is subjected to this operation at least twice more. The resulting solution is filtered through a Millipore filter, dialyzed against pyrogen-free distilled water and lyophilized.

In the column run, partially purified PRP in 20 mM phosphate buffer (pH at least 5.8) is applied to a column containing adsorbent hydroxylapatite equilibrated with 20 mM phosphate buffer, pH 5.8, and gradually eluted with a gradient of sodium phosphate buffer (pH 5.8). 100-mmo-vascular. The collected fractions are analyzed for pentose (and thus polyribosyl ribitol phosphate). The pentose-containing fractions are dialyzed against pyrogen-free distilled water and lyophilized.

(II) -Method for the preparation of a combination vaccine consisting of H. influenzae type b PRP and B. pertussis antigens to prepare a PRP vaccine lyophilized PRP (prepared as described above) is dissolved to a concentration of 20 mg / ml in phosphate-buffered sodium chloride solution (PBS) (0 g , 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, contain 0.01% thimerosal The sterile is sterile filtered through a 0.45 μm Millipore filter unit, filled into glass vials and stored at 4 ° C.

A concentrated solution of PRP is prepared by weighing a predetermined amount of polysaccharide and dissolving it in phosphate-buffered sodium chloride solution (0.113 g of potassium diphosphate, 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, containing 0f01% thimer). This concentrated PRP solution is then mixed with an appropriate volume of fresh B pertussis cell suspension to prepare a stock solution.

Characteristics of B. pertussis strain 138 are described in Bergeys Manual of Determinative Bacteriology, 8th Edition, Bushanan & Gibbons Editors, Williams & Wilkins Co., Maryland, USA. This strain is available from the American Type Culture Collection, Maryland, USA, under accession number 10380. The stock solution of the combination vaccine contains 200 pg PRP and about 70 opacity units (cr) cells per ml.

63674

The stock solution is maintained at 4 ° C for 90 days to detoxify pertussis antigens prior to preparation of the final product (vaccine) (containing 10 pg PRPr and 3.5 op units pertussis cells / 0.5 ml dose).

The invention is further illustrated by the following examples in which the temperatures indicated are in degrees Celsius.

Example 1 2.5 g of hydroxylapatite (e.g., Bio-gel HTP, Bio-Rad Laboratories, Richmond, CA) is added to 250 mL of a partially purified PRP preparation (after Cetavlon and ethanol treatment) (containing about 1.0 mg of PRP / ml) in 20 mM sodium phosphate buffer, pH 6.9, and the mixture is stirred in an ice-water bath (1-4 ° C) for one hour. The mixture is centrifuged in a Sörvall RC2-B centrifuge for 30 minutes at 16,000 g. The supernatant is then passed through a 0.65 μm millipore filter and this is repeated (each time treated with 2.5 g of hydroxylapatite) two more times. The resulting solution is filtered through a 0.65 and 0.45 Millipore filter, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product has strong immunogenic activity and contains very small amounts of impurities (such as nucleic acids, proteins, and endotoxins) (see Table I). About 170 mg of lyophilized PRP is obtained. The PRP yield by this method is about 70% based on the starting saccharide. The PRP must be stored under suitable conditions, such as 4 ° C in a desiccator over phosphorus pentoxide or silica gel.

Example 2

Partially purified PRP (300 mg PRP) is dissolved in 100 ml of 20 mM sodium phosphate buffer, pH 5.8 (3 mg PRP / ml). This solution is applied at room temperature to a column (5.0 x 45 cm) containing hydroxylapatite (ca. 250 ml pack volume) equilibrated with 20 mM sodium phosphate buffer, pH 5.8 and gradually eluted with 20 mM, 50 mM and 100 mM sodium phosphate buffer (pH 5.8). pH 5.8) with a gradient. The individual fractions (200 ml) eluted with 20 mM and 50 mM sodium phosphate buffer (pH 5.8) containing pentose (pentose assay by the original method) are collected, dialyzed against pyrogen-free distilled water and lyophilized.

7 63674

About 206 mg of lyophilized PRP is obtained.

The purity of the polysaccharide, PRP, is analyzed by determining the nucleic acids, proteins and endotoxins present as impurities. Protein content is determined by Lowry et al., J. Biol. Chem. 193: 265 (1951) using bovine serum albumin as a standard. Nucleic acids are determined by measuring the absorbance of the PRP solution at 260 nm. The absorbance of a solution of 50 μg of nucleic acid in 1 ml of water is measured in a cuvette through which the light passes 1 cm and is assumed to be 1,0. The co- (6) size of the molecules is evaluated by Sepharose ^ 4B or 2B gel filtration on a 1.5 x 90 cm column (Pharmacia-Fine Chemicals, Piscatway, N.J.). The partition coefficient (Kd) is determined from the elution model obtained in the original reaction (Herbert et al., Method in Microbiology, Vol. 5B, 285-291).

63674 8 n c

O I

• H CO

£ H W £ I 0) O £ £ -H d) <D r-l £ £ ·· rH «H £

£ V rH Λ -H

[0 W O &>, O O ^ rH / -> o tn S <

I £ O O O O) 0) CO

tn o o -H £ £ D

£ d- 1- bO £ tn

• H H <U \ ^ O H

C £ O v- ιΗ rH £ Λί • H £ Λί f ~ O £ £ £

H · Η Ή O * H O O

tn -Q H T- CQ £ Sm

rX dj X

O X Ή Pj 5 -H I O> i cm 0)

O O ^ CM HS

TD £ υ £ £ · Η C> 1 O -H £ CS "H Pj d) £ -HO) £ S>, • ho o o -h o) £ υ £ h

£ S "” £ £ £ · Η d) * H

· Η · Η OO Pj £ CQ fj ε tl) • H £ t— t— Ή £ Cl) · Η, £ £ 0) rH S £ S tn d) £ Cl)>, C <W> MM>, £ £ w · η - XHH · £ 0) £ H £: £ £> • H »H d) £ £ · γ j £ O, £ £ £ d) £ M * H £ * H £ £ ·ί · Η • H ch t ~~ £ <D £> <: Ä O d) r- ^ "*> M ίι £ X oo: £ · Η ld> £

, ¾ O ^ H d) ^ S £ i — I

£ £ ·· rH O tn O

| H Ph CQ: £ £ £ O

£ J- £ ^ Λ £ £ -W: £ Qh £ Λ

H QJ I: £: £ S d) £ H

£ 1 · Η H S Ph £ -P £

g £ Hl N -P

3 · Η (1) £ b0 £ r-v .h W <H oo oo SU d) T-p, - <- <φ o ^ Λ *> »0 Oj £ tn

£ rH P, dP OOH O rH H · O

C λ; Ρ, μ / .. £ o H v- S

· "£ £ CQ S r- £ oo § Z, £ cm O w · Η tn

Cl: £ · η W (1). * h <D H en tn 3 * h

M tn i en ·· * £ £ ζ, Q

m>, O CQ £ £ rH P £

A>, Otlf O 00 £ zf O £ -h £ cQ

• HiHH ^ "*> (fl £ <D · Η, £ H v_h H £ C toco, £ d) -H S TD Pj 7) H C d) · Η coco £ ω £ H £ O" 2 £ £ | <O. tn d) OPj £££ P £ H · OD £ £ H TD d) H £ SI £ -H -H £ £ Q e Φ: io: £ S £ S £ DCÖ £ J4>, XX Pj * HSH 0, ¾ I>, O ^ dJ d) · Η O tn £ H i WSS TD ^ Λ to en £ OH £ 0/3 • h tu s £ zr £ £ £> • O £

H rH £ m_ / O z ± KtJatJSPj'H £ "S

£ O * H i — 1 i — I · Η · Η X <C

£ 2 H o rHrHbOtntn co / 3

X: £: £ S dJ C * t- S

• h e e -s. £ o o) V) tn: £: £ Ph h · Η "£ >> £ HH« £ O ^ TDN: £ H + j h cm HHPj ”TDS ££ to

tn>,>, Oh £ £ £ QJ QJ

C Ή ·: £: £ W) 0h (D d) rH £ S

·· 6 SE ssa.Qj '-' E ^ rH.y

Oh rH · Η · Η · Η £ · Ρ £

Cm EC C / DC0 (/) it) e dj

Oh> WW £ TD O TD W 2 · Η £ 63674 9

Preparation of a combination vaccine

Example a A combination vaccine containing H. ingluenzae type b PRP and B. pertussis antigens was prepared as follows: 140 mg of PPP (prepared according to Example 2) is dissolved in 350 ml of sterile phosphate buffered saline (PBS) (0.113 g of potassium phosphate). 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, contain 0.01% thimerosal), and the solution is filtered through a 0.45 μm Millipore filter. This concentrated PRP solution (400 pg) ml) is used to prepare a combination vaccine containing PRP and B. pertussis antigens.

To 150 ml of concentrated PRP solution (400 pg / ml) is added 150 ml of a fresh B. pertussis cell suspension (strain 138) in PBS, pH 7.0 (turbidity 149 ECTS cells / ml) to prepare a stock solution of the combination vaccine. . This stock solution (300 ml) is kept at 4 ° C until the pertussis endotoxin level has decreased (at least 90 days). Sterility is tested with thioglycollate media (at 32-33 ° C). The pH of the thief toluene solution is measured 90 days after incubation and adjusted to pH 7.0-1. The final product (vaccine) used in animal experiments is prepared by diluting a stock solution of the combination vaccine with PBS to 10 μσ PRP and 3.5 cr. units of pertussis cells / 0.5 ml (dose).

The antibody response obtained with the combination vaccine in young rats is shown in Figures 1 and 2.

Example b A stock solution of PRP (400 pg / ml) is prepared by dissolving 30 mg of PRP (prepared according to Example 1) in 75 ml of sterile phosphate buffered saline (PBS), and the solution is filtered through a 0.45 μm Millipore filter. To 25 ml of this concentrated PFP solution is added an equal volume (i.e. 25 ml) of a fresh suspension of B. pertussis (strain 138) cells in PBS, pH 7.0 (containing 149 ECTS cells / ml) of the combination vaccine. to prepare a stock solution. This stock solution is kept for at least 90 days at 4 ° C. The sterility of the stock solution is tested as described above with thioglycollate media. The pH of the storage solution is 7.0-1. The final product used in animal experiments (vaccine) is prepared by diluting the stock solution of the combination vaccine with PBS to contain 10 pg PRP and 3.7 ECTS pertussis cells / 0.5 ml (dose).

10 63674

Example c

Prepare a PRP stock solution (200 pg / ml) in PBS (pH 7.0) as in Example b. A Pertussis stock solution (containing 75 op units / ml) is prepared by diluting 25 ml of a fresh B. pertussis cell suspension (149 op / ml). units) with an equal volume of PBS. Each stock solution is incubated separately at 4 ° C for 90 days or somewhat longer. The combination vaccine is prepared by taking 14 ml of PRP stock solution (200 pg / ml) and 14 ml (non-toxic) of pertussis stock solution (75 ECTS / ml) and diluting with 112 ml of PBS (final volume 140 ml). The finished vaccine used in animal experiments contains 10 pg PRP and 3.7 ECTS pertussis cells / 0.5 ml (dose).

Antibody formation in juvenile rats using the combination vaccine is shown in Figure 3 and Table II.

II

11 63674 3

Sh 3 rö CU + ->

CO CO., LTD

•B

«H C

3 3

•B

o CO

<o cu c \ (U 3 nj λ e

CHS

3: 3 -H

+ -> · π I <000 00: 3- LO LO 00 | i rs rs rs rs rs rs rs _ O 3 3 T-T-T-t- r ^ T-T- (β Ό o 3 T- v-

nj 'H -H

^ co -μ =

CU CU CO., LTD

i) ps * i) 3

O 1¾ 3 CU

u I Ή ft H CE O

ro 00 oc MX -H ~ ^ BX 3 -, ft · Η 3 Φ O · Η I — I · Η ο ^> ο -μ Μ ^ 3 c Ή ~: γΟ h COvhH ^ _ <μ H o 4j CU 6 Ή jj cu -μ m -μ ^ * O -H * «+»> ° '7? H Ή O ^ 3 I— | c -μ -μ CO CD CN Γ "r-OO · Η

S Λ! CO 00 LOJ-3-3- t ^ J-r- CO

O »· κ 3 O r— t— r— t— r— t- * c3- 03

, ¾ o CU · Η V-T-3CU

β 3 -μ 3 3 3 3 · μ X (3 · μ r-ί g 3 3 O 'Ο- CO CM Μ in r ^ 3 3 g I · ΓΟ CM Ot-J-OO O 00 LO (Ο-μ 3 5 33 h— t— h— t— o v- Ή ft H -μ -h v- co o rt CD CO Ai

n 3 3 3 -H

Tj> -ro CO 00 LO LO LOLDJ- + JCX

"ft I O T- T- O 00 00 OOJ-LO ft No.

Il CU fti T— T-T— T— T- T— t— CU 3

rt X x 3- CU

5 CL, -H 3 {rt I · Η o: 3 3 rt-H> cd oo o oo T-oocn: θ—

m -μ o σΐτ-r-o o cd v- X

WC x-v-v - ^ - ^^ O

<· Η X

3 CO X

rH fti -H

<—L »H ^ -ι · Η • μ I Cu I>

3 3 3 3 6 3 Λ Ό CU O

x ο-μο 30 ^ ^ ^ o ·> CU 3 ft 3 Ή (U 3 T. X X CO X ω X ooft r — C C 3 C 3 3 C \ -r — r w s- 'w LO 3

CU <ftC3-ft33 rfs • 'H -H

E II .ft OO 3 X

0 · η · η ^ w μ 3

, Υ ΟΌ h— / -m CD οί .H 3 O

1 ft -H x 3 Λ en · X · Η 3 ft · ^ ^ -μ + Jcsi μ co, Yoe ä ft * ex • H COi — I · Η Y 00 3 6 0-c I 3 3 co 3 γ 3 wp ^ .HOi-μ ·) —i co (¾ 6 3 oo to O, ft O 3 3 · η3 ω ^ '^ ω ^ + 3 3 ft ή μ + J · Η · Η + · Η ϋ ^ Μ μ 3 ft -ftSHCO —'o- ^ cO'- ': 1 3 3 μ 33 3 μ ιο κ ^ ίο · i <! Ο, ο ·· ft CU -μ 3 3 3 3 ^ - ^ - 3 ε ν Λ 0003 ιο ΐμ 3 ο μ μ · η γ, - m <- γ: CU Υ 3 3ft OhOh ft 3 Dh X Ο Ο 3 · Η 3 PS Ρί 3 3 Κ οο CUDS μμπ3 ftft 3 Λ Ο Ό

Claims (3)

12 63674 A method for isolating and purifying immunologically active polyribosyl ribitophosphate (PRP), a Haemophilus influenzae type b capsular polysaccharide, wherein the PRP is obtained by fermentation of Haemophilus influenzae type b strains under conditions known per se and the PRP is isolated under conventional conditions. as a hexadecyltrimethylammonium bromide complex in a manner known per se, characterized in that the PRP is purified by adding hydroxylapatite / 3 Ca3 (PO4) 2 Ca (OK) 2 · / 0.02-M in sodium phosphate buffer at a pH of about 6.7-6.9, stirring for about 2 At -10 ° C, centrifuging, collecting the supernatant and repeating the same procedure at least twice, filtering the supernatant with suitable filters, dialyzing against pyrogen-free, distilled water and lyophilizing. 2. A method for isolating and purifying immunologically active polyribosylribibli-______-phosphate (PRP), a Haemophilus influenzae type b capsular polysaccharide, wherein the PRP is obtained by fermenting Haemophilus influenzae type b strains isolated under conditions known per se, followed by PRP. by conventional ethanol precipitation and also in a manner known per se as hexadecyltrimethylammonium bromide complex, characterized in that the PRP is purified by column chromatography in 0.02 M sodium phosphate buffer at a pH of about 5.8 using hydroxylapatite / 3 Ca3 (PO2) 2 Ca (OH) 2 // eluting with a gradual gradient of 0.02-M and 0.05-M sodium phosphate buffer at a pH of about 5.8, isolating fractions by PRP analysis, dialyzing fractions free of pyrogen-free, distilled against water and lyophilization.