CA1272457A - Process for the extraction and purification of proteins from culture media producing them - Google Patents

Abstract

TITLE

Process for the extraction and purification of proteins from culture media producing them ABSTRACT

The process is applicable to the supernatant of engineered yeast cells disrupted in the presence of a non-ionic detergent: it comprises the precipitation of contaminants by polyethylene glycol and the treatment of this latter supernatant with either a bivalent metal cation or, after eventual ultrafiltration, with ammo-nium sulfate.

Description

~L~7;~7 TITLE
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Process for the extraction and purifica~ion of proteins from culture media producing them s Field of the invention~

The invention relates to an improved process for the extraction and purification of proteins from cul-ture media producing them: more particularly, the pro-10 cess relates to the extraction and purification ofhepatltis B virus surface antigen and alpha-l-antitryp-sin produced by recombinant DNA technique in engineered cell cultures, more particularly engineered yeast strains cultures.

Backaroun~d of the invention Differen$ microorganisms, among which bacteria and yeasts can be used as host organisms for different plasmids containing a DNA molecule comprising a nucleo-20 tide sequence coding for a ~pecific protein. Amongthese microorganisms, yeasts are presently preferred and currently used. For example, European Patent application publication No. 0 106 928 discloses the use oE SaccharomYce6 cerevisiae strains for the production 25 of hepatitis B virus surface antigen (HBsAg) and Euro-pean Patent application publication No. 0 103 409 rela-tes to the production of alpha-l-antitrypsin from yeast plasmids.

Protein production in yeast strainfi indeed pre-30 6ents sub6tantial advantages over production in bacte-rial strains. These advantages result from the rather easy growth of yeasts in large scale fermentors and from the fact that, contrary to bacteria, yeasts do appear to ressemble mammalian cells in their capacity ~2~724S7 ~o add carbohydrate groups to newly synthesi2ed pro-teins.
Navertheless the extraction and purification of the protein from a yeast culture do pre6ent technical 5 problems due to the rather complex chemical composition of the yeast cell and more particularly to the presence of high lipid levels when the yeast growth is extended in order to improve the polypeptide production yield.
For example, the Saccharomyces cell wall is 10 thought to consist of 3 layers : ta) an inner layer of alkali-insoluble B-glucan, (b) a middle layer of alkali-soluble B-glucan and (c) an outer layer of gly-coprotein in which the carbohydrate consi6ts of phos-phorylated mannan; beneath the cell wall is a cytoplas-15 mic membrane consisting of a very complex mixture ofneutral lipids (mono-, di- and tri-glycerides), free and esterified sterols, a complex sphingolipid, glyce-rophosphatides and neutral as well as acidic glycoli-pids; the nucleus contains DNA, various species of RNA
20 and a polyphosphate; vacuoles may contain a great variety of components of both high and low molecular weights, the~ 6erve as storage vesicles for a number of hydrolytic enzymes; the mitochondria are rich in lipid, phospholipid and ergosterol components of the membrane 25 system and the cytoplasm contains a.o. large quantities of ribosomes, polyphosphates, glycogen and a number of glycolytic enzymes. Most yeast cells (such a6 Saccha-romyces species) al60 contain some amount of lipid in the form of globule6 the amount of which does increase 30 in extended cultures.
A number of multi-steps processes have been dis-closed for the extraction and purification of proteins from different sources. Examples refeLring to proteins produced by engineered microorganisms are those publi-35 shed by Th. STAE~IELIN et al. (J. Biol. Chem. 256;

~2D~;7 9750-54; 19~1), K. MURR~Y et al. (The EM~0 J.
l:p, 645-650: 1984) and R.A. HITZEMAN et al. (Nucl. Ac.
Res. 11: 2745-2763: 1983).
Typically, when the produced protein is cell 5 bound, those different processes involve 3 series of s~eps.
In the first eeries of steps, the desired protein i~ re~oved from the cell interior. Therefore, the cells are either lysed (e.g. by enzymatic treatment) or 10 disrupted (e.g. by mechanical forces such as shearing forces, e.g. X-press or French pres6) or shaking with glass beads, eventually with addition of a detergent (see for instance K. MURRAY et al. loc. cit. and R.A.
HITZEMAN et al. loc. cit.).
In the second series of steps, the medium is enri-ched in desired protein, e.g. by fractional precipita-tion by addition of ammonium sulfate and/or in the pre-sence of polyethylene glycol.
Finally, in the third series of steps, substan-20 tially all contaminants are eliminated from the medium, e.g. by one or several operations selected from the group comprising ultrafiltration, ion exchange, gel filtration chromatography and isopycnic gradient cen-trifugation.
In such process, it is obvious that contaminants like accompanying proteins (cited by R.A. HITZEMANN et al. loc. cit.), nucleic acids and lipids and more par-ticularly high lipid levels have a harmful influence on at least one of the steps of the third series (e.g.
30 ultrafiltration and column chromatography) and the pro-teolytic enzymes must be eliminated rapidly from the medium. ~oreover, it has also been noticed that ammo-nium sulfate precipitation is not possible without a previous rough delipidation because lipids interfere 35 with this precipitation.

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Therefore it is of prime importance to dispose of a method wherein most of the contaminants are elimina-ted before the third seLie6 of steps.
Soma among the previously described processes dis-5 close the use of polyethylene glycol as a selective precipitating agent for proteins and a method for the precipitation of lipoproteins from plasma by using lipoprotein-polyanion-metal interactions has also been reported.
The method for fractional precipitation of pro-teins by using nonionic water-soluble polymers, in par-ticular polyethylene glycol (PEG) has been introduced by POLSON et al. (Biochem. Biophy6. Acta 82; ~63-475;
1964) and discussed by different authors. Among them 15 W. HONIG et al. (Analyt. Biochem. 72; 502-512: 1976) mention that "the specificity of precipitation, that is the ratio of desired protein and tot-al protein, can be improved by using PEG fractions of lower average mole-cular weight than the generally employed PEG 6000".
20 Nevertheless although "by manipulation of pH concentra-tes of individual plasma proteins may be obtained" the authors added "however, purification of more complex protein mi~tures such as the supernatant of a cell homogena~e is considerably poorer".
P.R. FOSTER et al. (Biochim. Biophys. Acta 317;
505- 516; 1973 have described a method for the precipi-tation o~ enzymes from cell extracts of SaccharomYces cerevisiae by PEG. Methods for the concentration and purification of viruses and bacteriophages with PEG
30 have been disclosed by B.P. VAJDA (Folia Microbiol. 23, 88-g6; 1978) and G.J. LANCZ (Arch. Virusforsch. 42;
303-306; 1g73). In the field of hepatitis antigen iso-lation, the purification of hepatitis B surface antigen (HBsAg) by a method comerising two successive treat-35 ments with PEG 6000 has been described by Ph. ~DAMOWICZ

~2~ 7 et al. (p. 37-49 INSERM SYMPOSIUM No. 18, HEPATITIS B
VACCINE, Publ. ELSEVIE~, Amsteedam, Holland, 1981). In this method of HBsAg purification from plasma, immune complexes and most of the lipop~oteins are, in a first 5 step, precipitated from serum by PEG 6000 at a concen-tration of 5.5 % and, in a second step, HBsAg i8 preci-pitated from the isolated supernatant by addition of PEG at a final concentration of 10 %.
In the patent literature, 10 - US Patent 3 790 552 discloses a method for removing hepatitis-associated antigen from a protein fraction which comprises a step wherein PEG having a molecular weight 200-6,000 is used in an amount of 12-30 (w/v) for precipitating said antigen.
15 - US Patent 3 951 937 discloses a process for the puri-fication of hepatitis B antigen (HBAg) involving a double precipitation of HBAg with PEG (4.0-4.5 weight percent) having a molecular weight of at least 600.
- US Patent 3 994 870 discloses a method for purifving hepatitis B antigen (HBAg) wherein HBAg is precipita-ted by addition of 4.0-4.5 weight percent PEG and thereafter subjected to affinity chromatography uti-lizing insoluble concavalin A as a chromatographic adsorbent.
25 - European patent application, publication No. 0 112 506 discloses a process for producing a hepatitis B infection preventiny vaccine from plasma comprising ammonium sulfate precipitation followed by adsorption on colloidal silicate and two successive precipitation steps with PEG (having a molecular weight of 2,000 - 10~000) at a 3-7 % (w/v) to preci-pitate hepatitis B virus and immune complexes and at a 15-20 % (w~v) in the supernatant to precipitate HBsAg.

- In the field of alpha-l-antitrypsin isolation, Japa-ne~e patent application 912B-335 filed July 24, 1984 and assigned tD Fuji Re~ioI~ (~W~It abskract 84~217127) discloses the precipitation of alpha-l an~psin fram pl~ fraction by addi~on of PEG in an am~unt of 15-20 % (w/v~.
As mentioned above, a method for the precipitation of lipoproteins by u~ing lipoprotein-polyanion-metal interactions has also been previou~ly repo~ted (for in~tance : M. BURSTEIN et al. Adv. Lip. Res. 11: 67-10 108; 1973 and A. VAN DALEN et al. Biochim. et Biophys.Acta 147;421-427: 1967~. This method iE performed by interaction between the lipoproteins, a bivalent metal cation and an acidic poly6accharide and, in the6e ope-~ative conditions, the amount of precipitate i~ a func-15 tion of the bivalent metal cation concentration in themedium.

Description-of the invention The ~tarting material ~or the process of thi6 20 invention (herein also referred to a6 'crude extract~) i~ the supernatant of engineered yea6t eells having produced a cell-bound protein and di6rupted in the pre-sence of a non-ionic detergent, a6 well known in the art.
According to the pre~ent invention, there is then made a combination of the fractional precipitation by PEG ~hereaftec refered to as first step) followed by either a fractional precipitation by polyvalent metal cation (more particularly bivalent metal such as cal-30 cium and mangane6e) or by ammonium sulfate treatment, said ammonium ~ulfate treatment being eventually prece-ded by ultrafiltration of the supernatant from the first 6tep.
The present invention derives from the diEcovery 35 that, when solid PEG (e.g. PEG 6000) is employed at a concentration of 6-12 % (w/v) for the extraction of HBsAg from engineered yeast cells disrupted in the pre-sence of a nonionic detergent, HBsAb does not precipi-tate. Moreover, by subæequent addition of ammonium 5 sulfate, a two phase sy~tem is formed which is charac-terized by the fact that the HBsAg is present in the PEG phase while most contaminants ace in the aqueous pha~e. This result is surprising because, according ~o the general teaching of the prior art in other crude 10 media, these operative conditions should provoke the precipitation of the BHfiAg. Thus, it is a first object of the pre6ent invention to use PEG as a solvent for the desired protein produced by yeast cell cultuLes whereas most of the contaminants are eliminated by pre-15 cipitation from the medium.
According to molecular weight, PEG does exist either in solid form or in liquid form, the frontier between both forms being around a molecular weight of 1500 .
In the f iLSt step of the process according to the invention (which could also be considered as a clarifi-cation step), PEG6 having different molecular weights can obviously be used with adequate concentration adjustment according to the corresponding molecular 25 weight.
For instance, the PEG concentration is preferably 6 to 12 % tw/v) when using solid PEG (e.g. PEG 6000) and from 10 to 35 % tv/v) - and preferably from 20 to 30 % (v/v) when liquid PEG (e.g. PEG 300 or 400) is 30 employed. Nevertheless, the use of liquid PEG is pre-; ferred for technical reasons among which the easiness of later ultrafiltration.
Thus, according to the invention, there is provi-ded a process for the extraction and purification of 3~ proteina, mo~e particularly ~3aA~3 or alpha-l-antitryp--` ~3L2~

sin, from the supernatant of yeast cells disrupted in the presence of a nonionic detergent (preferably a polysorbate detergent), said supernatant being herein refereed to as 'crude extract~, which method comprises 5 a first step wherein either 6- 12 % (w~v) of solid PEG
or preferably 10-25 % (v/v) of liquid PEG is added to the crude extract brought to pH 6 (+ 0.1), the above limits of PEG concentrations being mainly fixed by the point at which clarification is reached since further 10 addition of PEG has only detrimental effects, i.e.
higher viscosity of the medium and risk of undesired co-precipitation of HBsAg or alpha-l-antitrypsin.
In the process of the invention, P~G i6 regarded as a polymerized oLganic solvent favouring a. o. tha 15 precipitation of the (lipo)proteins whose isoelectric point is close to pH 6 and the solubilisation of the other (lipo)proteins, i.e. those which have a markedly different isoelectric point and those which are highly hydrophobic.
It has been noticed that this PEG clarification step precipitates about 75 % of the contaminant pro teins, 90 % of the polysaccharides, 94 % of the nucleic acids and 45 % of the lipids, while substantially the whole HBsAg or alpha-l-antitrypsin content i6 recovered 25 in the supernatant.
As indicated hereinabove, the present invention includes a combination of steps, the first one of them being a clarification step, to yield a partly purified and ev0ntually somewhat opalescent solution of the 30 desired protein.
In the second step of the process of the inven-tion, the obtained partly purified solution is either treated with a polyvalent - more particularly bivalent - metal cation such as an aqueous solution of calcium 35 or manganese chloride at a final concentration of 30 mM

~4~7 g or treated with ammonium sulfate and this ammonium sul-fate treatment i8 performed either after ultrafiltra-tion of the opalescent solution by addition of solid ammonium sulfate up to ~0-50 % ~aturation, according to 5 the classical salting out method to precipitate the desired protein which can be taken over in a phosphate buffer or by addition of ammonium sulfate up to ~0-50 %
saturation to the PEG containing supernatant, forming a two-phase system with the desired protein in the PEG
10 phase.
; Each of these variations for the second series of steps does provide a clear solution of the desired pro-tein substantially purified regarding its polysacchari-des, nucleic acids, lipids and contaminant proteins 15 content.
The so-obtained solution can then be processed in the above defined clas6ical third series of steps, e.g.
eventual ultrafiltration, gel filtration and column chromatography and, in a preferred embodiment of the 20 present invention, this third series of steps comprises successively an ultrafiltration, a first gel filtra-tion, a column chromatography on weakly alkaline anion exchanger with diethylaminoethyl (DEAE) groups and a second gel filtration or a cesium chloride gradient 25 centrifugation to yield a highly pueified ~BsAg or alpha-l-antitrypsin solution suitable for medical use.
When tested by polyacrylamide gel electrophoresi6 (sodium dodecyl sulphate), the product recovered after the second gel permeation is shown to be more than 9~ %
30 pure with regard to the 23 K band which i6 characteris-tic of HBs~g of yeast origin.
Thus, the primary advantage of the method of this invention is that it does remove substantially all polysaccharides, nucleic acids, lipids and proteina-35 ceoua material and another advantage ot the invention ~, is that the desired protein may finally be separated in relatively high yield.
When the process of the invention i6 applied to a crude extract of HBsAg from engineered yeast cells, the 5 obtained HBsAg i~ bound to the non ionic dete~gent for-ming therewith composite micelles of a diameter of about from 17 to 20 nm and it ha6 been found that the ratio expres6ing the amount of non ionic detergent on the amount of proteins, total lipids and polysorbate in 10 the polysorbate composite micelle is from 15 to 35 %
(w/v) when the assays are performed by colorime~ric method for proteins (LOWRY), total lipids ~ZOLLNER) and non ionic detergent (HUDDLESTON and ALLRED).
The composite micelle~ obtain0d by the process of 15 the invention by using a polysorbate a non-ionic deter-gent are novel compounds which are also an object of this invention: they are immunogenic and can be formu-lated into vaccine form like classical ~B6Ag as it is well known in the art for hepatitis B virus vaccine 20 preparation.
The invention jis illustrated by the following examples which are not limitative of the scope of the invention.

ExamPle 1 Pelleted yeast cells (3850 g) of an engineered yeast 6train expressing HBsAg which have grown up to 30 g dry cells weight per litre of culture are suspen-ded in 7.12 litres of Na2HP04 solution (7.098g/1).
This suspension i6 supplemented with 1~2.5 ml ~ %
(w/v) EDTA solution, 38.5 ml polysorbate 20 and 385 ml isopropanol containing 2.7 g phenylmethylsulfonyl fluo-ride (PMSF). The pH is adjusted to 8.1 (+ 0.1) with NaOH (10 % w/v in water). The suspen6ion is refrigera-35 ted in an ice bath and diarupted by Z paasagea th~ough a cooled gla~ beads homogenizer. The homogenate (crude extract) is then centrifuged foc 30 minutes at 13000 ~.
PEG 400 (2,5 1) i~ ~lowly added wieh ~tirring ~o 5 the ~rude extract (7.7 1) which is maintained below 15 C. The pH i6 then adju~ted to 6 (~ 0.1) by addi-tio~ of acetic acid (5 M~ and the medium i~ ~tored for one hour at 4 ~C before being centrifuged at 7,400-11,000 g for 45 minutes.
The supernatant i~ brought to pH 7.0 (+ 0.05) with N NaOH and 300 ml of M CaC12 ~i.e. 30 ml per liter of 6upernatant) i8 added thereto. The pH iB readju~ted to 7 (_ 0.05~ with N NaOH and stored overnight at 4 C.
The ~uspension i8 centrifuged at 3600 g.
15 for 45 minutes and the supernatant i~ ultrafiltered in an AMICON DC (apparatus ~old by AMICON CORP., DANVERS, MA, USA)) equipped with a cartridge having a cut-off of 100,000 dalton6 and thereby ~irst concentrated up to 1/4 (1500 ml) of it6 initial volume. The solution i6 20 then washed csntinuou61y with 12.5 1 of 10 mM trometa-mol adjusted to pH 8 (~ 0.1) by addition of N hydro-chloric acid (thi~ buffer i6 hereafter referred to as trometamol pH 8) and ~upplemented with PMSF (l~M), i60-propanol (2.5 % v/v) and EDTA (2 MM) (final concentra-25 tions). The retentate i6 then furt~er concentrated to3S0 ml.
The concentra~ed solution iB applied to a column (0 10 cm x 100 cm) ~ontaining 7 1 o~ FRACTOGEL
TSK HW65(F) (a semi-rigid gel consi6ting of hydrophilic 30 vinyl polymer6 with numerous hydroxyl groups on the matrix 6urface with particle 6ize 32-63 um manufactured and sold by E. ~erck~ Darmstadt, FRG) equilibrated in trometamol/HCl buffer 10 mM pH 7 (~ 0.01) supplemented with 5 % (v/v) ethylene glycol.
35 * Trade mark

2~7 The HB6Ag antigen containing peak is applied to a column (0 5 cm x 30 cm3 of 300 ml of FRACTOGEL
TSK DEAE 650 (M) (a weakly basic anion exchanger wherein diethylaminoethyl groups are bound to the 5 hydroxyl groups of FRACTOGEL TSK HW-65 mateix via ether linkages, manufactured and fiold by E. Merck, Darmstadt~
FRG) equilibrated in trometamol pH 8 at 4 C . The column is washed with trometamol pH B containing 0.05 M
NaCl and HBsAg is eluted with 0.15 M NaCl in trometamol 10 pH 8.
The eluate is applied to a column of FRACTOGEL
TSK HW65(F) equilibrated with Na2HP04/NaHzpo4 buffer (10 mM~ pH 6.8 supplemented with NaCl (150 mM) yielding a solution of HBsAg/polysorbate composite 15 micelles.
The purification level obtained at each stage of the purification is shown in Table I.

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TABLE I
.. . . ................... . .. _ Fraction Vol Total Total Total Total Total (1) HB6Ag Proteins Polysac- Nucleic lipids by RIA charides acids (mq) tq~ (q) (mq2-- (q2 Crude extract7.7 1232 259 114 32500 104 PEG
- 10 supernatant 8.02 1100 64 9.5 641 53.7 CaC12 supernatant 8 1190 37 3.8 512 27.2 Retentate .350 1320 26.7 .744 37.6 3.596 HBsAg peak 15 TSK 1.06 736 .935 .014 16 .352 Eluate TSK-DEAE.15 722 .428 .00842.6 .275 HBsAg peak TSK _ 45 1117 244 _ 0094_ 2 .179 20 RIA : Radio Immuno Assay The following Table II summarizes the composition of composite micelles of 3 different vaccine batches obtained by the above peocess with polysorbate 20.
TABLE II
.
Batch Proteins Total Polysorbate 20 Ratio Lipids C
(ug/ml) (ug/ml) (ug/ml) ~/B/C
(A~ (B) (C~ __ I 20 20 8.5 18 ~: II 20 16 8.4 19 III 20 14.5__ _ 15.6 _ _ 31 -~2~

Exam~le 2 PEG 400 (a82 ml) is slowly added to 2,650 1 of crude extract prepared as de~c~ibed in example 1 and the pH i~ adjusted to 6. (+ O.l). The medium i~ main-5 tained at 4~ C for one hour and then centrifuged at7400 g. The ~upernatant is concentrated to 1000 ml on an AMICON DC equipped with a cartridge having a cut-off of 100,000 dalton~ and then washed with 3 ~olume~ of 20 mM trometamol pH 8. Powdered ammonium sulfate 10 (277 g) i~ then added slowly to the retenta~e, under stirring and at 4 C. After 1 hour at 4 C, the preci-pitate i6 centrifuged for 15 min. at 1000 g. The 6upernatant i6 di6carded and the precipitate is di6sol-ved in 400 ml 20 mM trometamol pH 8 supplemented with 15 1 mM P~SF. The solution i6 ultrafiltered on an Amicon DC equipped with a cartridge having a cut-off of 10 dalton6. The retentate (500 ml) i6 washed ~ith 2 volu-me6 of trometamol pH 8 and then applied to a column containinq 300 ml of TSX~DEAE 650 M gel equilibrated 20 with trometamol pH 8. When the pa6sage of the sample i6 completed, the column i6 washed with 0.05 M NaCl in trometamol pH 8 and a linear gradient of NaCl (0.05 M -0.5 M) i6 then applied to the column. The fraction eluted with 0.15 M NaCl contains the HB~Ag; it i6 con-25 centrated to 50 ml in an Amicon DC equipped with a car-tridge having a cut-off of 10,000 and applied to a column (50 x 100 cm) of Sepharo6e*4B-Cl (an agaro6e gel manufactured and sold by Pharmacia Fine Chemicals, Upp-sala, Sweden) equilibrated with 20 mM t~ometamol pH 8 30 supplemented with 5 % ethylene glycol, yielding a solu-tion of HB6Ag/poly~orbate compo6ite micelles.
The purification level obtained at each stage i~
6hown in Table III.
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TABI.E III

Fraction Vol Total Total Total Total HBsAg Proteins Poly~ac- ~ucleic by RIA charides acids __ (ml) ~m~? ~q~ _(q~
Crude extract 2650 188 114.721 36.9 9250 PEG supernatant 2530 160 22.7553.321 536 ~etentate 105 1000 150 7.468.181 175 - 10 ~MS-pellet 40080.8 1.248 .0248160 Retentate 10 100072.4.529 .029 8~
Eluate TSK-DEAE 560 44 .157 0.073 3 HBsAg peak SePharose 4B-CL 200 23 030 0003 05 15 RIA : Radio Immuno Assay I Example 3 ! , HBs~g crude extract (2 litres) is clarified by PEG
400 as described in example 1. Powdered ammonium sul-- 20 fate (AMS) (450 g) is added~ thereto under stirring (final concentration is 45 ~ AMS saturation). After solubilisation of the ~MS, the solution is kept at ~ C
for 3 hours at the end of which period two distinct phases are obtained which are separated in a separation 25 funnel : a clear (yellow) upper phase (PEG ~00 phase) and a clear lower phase (aqueous phase), each phase representing about + 50 ~ of the original volume. The aqueous phase is discarded and the upper phase is ; ultrafiltered on a Amicon DC as described for the 30 CaC12 supernatant in example 1. The purification procedure is then further carried out as described in I example 1 yielding a solution of HBsAg/polysorbate com-I posite micelles.
! The purification level obtained at each stage is 35 shown in Table IV.

~7 TABLF IV
.
Fraction Vol Total Total TotalTotal HBsAg Proteins Polysac- Nucleic by RIA charides acid~
(ml) (mq~ (q) (ql (mq2 ~ = _ Crude extract 200099 55 22 55~8 PEG supernatant 200092 13.25.68 243 PEG phase 1000 98 . 2.8 1.536 98 10 Retentate 50 98 1.5 .146.66 HBsAg peak TSK 123 52 .288.0077 2.84 Eluate TSK-DEAE Z5 54 .0377.0011 .44 HBsAq peak TSK 60 35 0207.00154 _ 39 RIA : Radio Immuno Assay Example 4 HBsAg crude extract (2 lîtres) is clarified by PEG
400 as described in example 1. Powdered ammonium sul-fate (AMS) (450 g) is added thereto under stirring 20 (final concentration is 45 % AMS saturation). After solubilisation of the AMS, the solution is kept at 4 C
for 3 hours at the end of which period two distinct phases are obtained which are separated in a seearation funnel : a clear (yellow~ upper phase (PEG 400 phase) 25 and a clear lower phase (aqueous phase), each phase representing about ~ 50 % of the original volume. The aqueous phase is discarded and a one litre aliquot of PEG phase (upper phase) is diluted twofold with 10 mM
trometamol pH 8 and applied to a column containing 30 300 ml of FRACTOGEL TSK-DEAE 650(M) gel equilibrated with trometamol pH 8 at a ~flow rate of 100 ml/hour.
The gel is washed with trometamol pH 8 containing 0.05 M NaCl and the HBsAg is then eluted with a NaCl gradient (0.05 - 0.5 M NaCl in trometamol pH 8). A
35 HBsAg peak is eluted with 0.15 M NaCl and, after con-I

centration in an AMICON DC equipped with a cartridge having a cut-off of 10.000. the retentate i6 applied to a column (5 x 100 cm) of Sepharose 4B-Cl equilibrated with trometamol pH 8 containing 5 % tv/v) ethylene gly-5 col, yielding a peak of HBsAg~polysorbate compositemicelles.
The purification level at each stage i6 shown in Table V.

TABLE V

Fraction Vol Total Total Total Total HBsAg Proteins Polysac- Nucleic by RIA charides acid~
(ml) (ma~ (q) (q) (mq) Crude extract 2100200 60.600 lg.ooo 4R60 PEG phase1200 2103.100 1.436 80 Eluate TSK-DEAE 460 134 .750 .0048 05 20 HBsAg peak SePharose 4B-CL 150 110 120 0014 0.3 RIA : Radio Immuno Assay Example 5 HBsAg crude extract (500 ml) is clarified by slow addition of a solution of 50 g PEG 6000 in 160 ml of water under stirring at 4. The pH is adjusted to 6.1 by addition of 5 M acetic acid. After a one hour sto-rage at 4, the extract is centrifuged for 15 min. at 30 7000 g. Powdered ammonium sulfate (157 g) is then added to the~supernatant under stirring (final concen-; tration is 50 % in saturated ammonium sulfate). After solubilisation, the solution is kept at 4 C for

3 hours, after which period two distinct phases are 35 obtained which are separated in a separation iunnel.

~2~7 The upper phase (PEG phase containing the HBsAg) is diluted twofold with trom0tamol pH 8 and then applied to a column containing 300 ml of TSK-DEAE 650(M) gel equilibratad in trometamol pH 8 at a flow rate of 5 100 ml/hour. The gel i8 wa~hed with trometamol pH 8 containing 0.05 M NaCl and the HBsAg antigen is eluted with trometamol pH 8 containing 0.15 M NaCl, concent~a-ted in an AMICON DC equipped with a cartridge having a cut-off of 10,000 and the ~etentate is then applied to 10 a 2 litres column (5 x 100 cm) of FRACTOGEL TSK-HW65 (F) equilibrated with trometamol pH 8 containing 10 %
(v/v) ethylene glycol, yielding a solution of HBsAg/
polysorbate composite micelles.
The purification level at each stage is shown in 15 Table VI.

- TABLE VI
. _ _ FractionVol Total Total Total Total HBsAg Proteins Polysac- Nucleic by RIA charides acids ; (ml) (mq) tq) (q) (mq) Crude extract 500 123 Z2.6006.400 1200 PEG supernatant 490 100 4.153.509 24 25 PEG phase 270 110 1.820.2096 5.3 Eluate TSK-DEAE 150 66 .258 .028 .4 ElBsAq peak TSE~ 60 _ 68 065 00048 084 RIA : Radio Immuno Assay 0 ExamPle 6 PEG 400 (13 ml) is added slowly to alpha-l-anti-trypsin crude extract (120 ml) from an engineered yeast culture and the pH i6 adjusted to 6.1 with 5 M acetic acid. After a one hour storage at 4 C, the medium is 35 ~entri~uged a~ 5000 g f or 15 minutes an3 1 U CaClz ~LS7 solution is added slowly to a final concentration of 40 mM. The pH is adjusted to 7 with 1 U NaOH and, aftec overnight storage at 4~ C, the medium is centri-fuged a~ 7000 g for 15 minutes. The supernatant is S diluted twofold with 50 ~M trometamol adjusted to pH 8.7 by addition of 0.1 N hydrochloeic acid and applied to a column of FRACTOGEL TSK-DEAE 650(M) (20 ml) equilibrated with 50 mM trometamol pH 8.7 pre-pared as indicated above but supplemented with 5 mM
10 mercaptoethanol. The column is washed with trometamol pH 8.7 as indicated above but supplemented with 10 mM
NaCl, a NaCl gradient is applied (10 mM - 250 mM) and the alpha-l-antitrypsin is eluted at a NaCl concentra-tion of 124 mM, separated from a protein peak eluted at 15 80 mM NaCl. The alpha-l-antitrypsin peak is concentra-ted in an ~MICON DC equipped with a cartridge having a - cut-off of 10,000 and applied to a Sephadex 150 column equilibrated in 50 mM trometamol pH 8.7 prepared as indicated above, yielding a solution of purified 20 alpha-l-antitrypsin.
The purification level obtained at each stage is shown in Table VII.

TABLE VII
Fractlon Vol Total Total Total AAT Proteins Polysac-by ELISA charides (ml) tmq~ _ (q) _ (q) 30 Crude extract 120 1804.018.538 PEG supernatant 100 2121.300.290 CaC12 supernatant 100 194.660 .110 Eluate TSK-DEAE _ 520 180 _ 210 0052_ AAT : alpha-l-antitrypsin ~z~
- ~o -ExamPle 7 .
The technique i8 as de~cribed in Example 1 but 300 ~1 of ~ ~nC12 are sub~tituted for the 300 ml of M
CaC12 therein specified. The characteristic6 of the 5 final product are similac to tho~e of the product obtained at the end of Example 1.

Example 8.
The technique i8 as described in Example 1, but 10 20 ml of Triton ~-lOO*(a product manufactured by Rohm and Haas: Darm~tadt, FRG) are substituted for the 3~.5 ~1 poly60rbate 20 therein specified. The charac-teristics of the final product are similar to those of the product obtained at the end of Example 1.

ExamPle 9 The technique i6 as described in Example 1, but 38.5 ~1 of poly60rbate 80 are sub6tituted for the 38.5 ml of polysorbate 20 therein 6pecified. The cha-20 racteristics of the final product are 6imilar to tho6eof the product obtained at the end of Example 1.

Example 10 The solution of HB~Ag composite micelle obtained 25 at the end of Example 1 is adju~ted to a protein con-tent of 10 ug per millilitre by addition of NaCl, pho~-phate buffer (Na2HP04~NaH2P04) and ALHYDRO-GEL (an aluminium hydroxide gel manufactured and sold by SUPERPHOS Export Co, Copenhagen, Denmark~ up to 30 ~inal concentrations of 0.9 % (wJv), 20 mM and 0.15 %
(w/v) of Al(OH)3, respectively, the final pH~ being 6.9.
The preparation i6 sterilized and distributed into 2 ~1 gla6s vials, each containing one ml dosage unit of 35 vaccine.
* Irade mark ,',~

~2~

Example 11 Dosage units of the vaccine of Example 10 have been administered by in~ramuscular route to two series of seronegative subjects in 2 succe6sive injections, at 5 a one month interval. Although these injections should be consideced as priming administration which should be followed by a booster, e.g. 2 months after the first injection. positive antibody respon~es were already noticed one and two months respectively after the fir~t 10 administration.
In the first series (comprising 32 seronegative subjects) one month after the fir6t administration, the seroconversion rate was 20/32, i.e. 62.5 % with a geo-metric mean titre (GMT) of g.95 milli international 15 units (MIU) and one month after the second administra-tion the seroconversion rate was 31/32 i.e. 96.9 % with GMT 36 MIU.
In the second serie6 (comprising 46 seronegative subjects) one month after the first administration the 20 seroconversion rate was 31/46, i.e. 67.4 % with GMT
13/6 MIU.
Recording of clinical signs and symptoms revealed no temperature rise and no noticeable local reaction among the vaccinees.

I .

Claims (17)

What we claim is:

1. In a method for extracting and purifying a cell-bound protein from the supernatant of engineered yeast cells having produced said protein and disrupted in the presence of a non-ionic detergent, a process which comprises adjusting the pH of the supernatant to 6 (?
0.1), adding thereto either liquid or solid polyethylene glycol up to clarification of said supernatant and treating the clarified supernatant either with a bivalent metal cation or, after eventual ultrafiltration, with ammonium sulfate for separating said protein.

2. A process according to claim 1 wherein liquid polyethylene glycol is added at a final concentration comprised between 10 and 35% (v/v).

3. A process according to claim 1 wherein solid polyethylene glycol is added at a final concentration comprised between 6 and 12% (w/v).

4. A process according to any one of claims 1 to 3 wherein the protein is hepatitis B surface antigen.

5. A process according to any one of claims 1 to 3 wherein the protein is alpha-1-antitrypsin.

6. A process according to claim 1 wherein the clarified supernatant is treated with a bivalent metal cation.

7. A process according to claim 6 wherein the bivalent metal cation is calcium or manganese cation.

8. A process according to any one of claims 6 and 7 further comprising separation of the precipitate, ultrafiltration of the solution, gel permeation of the retentate, ion exchange chromatography of the protein containing peak and, eventually, a second gel permeation or a cesium chloride gradient centrifugation.

9. A process according to claim 1 wherein the clarified supernatant is treated with ammo-nium sulfate up to 40-50 % saturation and isolating the purified protein in the polyethylene glycol phase.

10. A process according to claim 9 wherein the polyethylene glycol phase is further subjected to even-tual ultrafiltration followed by gel permeation and ion exchange chromatography.

11. A process according to claim 9 wherein the polyethylene glycol phase is further subjected to even-tual ultrafiltration followed by ion exchange chromato-graphy and gel permeation.

12. A process according to claim 2 wherein the clarified supernatant is subjected to ultrafiltration and the retentate is treated with ammonium sulfate up to 40-50 % saturation in order to precipitate the pro-tein which is taken over in an adequate buffer.

13. A process according to claim 12 further com-prising ultrafiltration, gel permeation and ion exchange chromatography.

14. A method according to claim 1 wherein the non-ionic detergent is a polysorbate.

15. A method according to claim 14 wherein the polysorbate is polysorbate 20.

16. Hepatitis B surface antigen/polysorbate compo-site micelle containing from 15 to 35 % (w/w) of poly-sorbate.

17. Hepatitis B vaccine containing as active ingredient a composite micelle according to claim 16.