DD286721A7 - PROCESS FOR OBTAINING PROTEIN A - Google Patents

Abstract

The invention relates to a microbial method for obtaining protein A from culture filtrates of staphylococci or other, preferably recombinant bacterial protein A producers. Its field of application lies in technical microbiology; The field of application of the protein produced according to the method is biotechnology (eg isolation of antibodies) as well as human medicine (therapeutic, diagnostic). The object of the invention is to provide highly purified protein A for the various applications mentioned. The object associated with this aim is achieved by submerging a bacterial strain capable of carrying out protein A biosynthesis in a manner known per se and separating its biomass from the culture solution following fermentation, if appropriate after cell disruption Then resulting culture supernatant with a wide-pore silica gel (pore gas desalt between 10 nm and 50 nm) and treated from the loaded adsorbent, optionally after a washing step, the target protein at extreme p H values with solutions of monohydric or polyhydric alcohols, ketones, amines or eluted from amides and isolated from the resulting eluates by ion exchange chromatography as a highly purified product. {Protein A; Culture supernatant of a submerged fermentation; Treatment with far-pored silica gel; Elution with solutions of alcohols, ketones, amines or amides; highly purified product; Biotechnology; Human Medicine}

Description

See 1 page Figure 1

Field of application of the invention

The invention relates to a method for obtaining pure protein A from culture filtrates of staphylococci or other, preferably recombinant bacterial protein Α producers. Protein A is a cellular protein naturally produced by many staphylococci which is capable of specifically binding IgG-type immunoglobulins to their respective Fc (fragment-crystalline) portion. Protein A is used in serological diagnostics alo labeled probe (enzyme, fluorescence or radioactive label) for the detection of specific antibodies. Further applications are the possibility of isolating antibodies, also monoclonal, by covalently bound protein A to insoluble carriers. There are also approaches to the therapeutic use of carrier-fixed protein A in autoimmune diseases.

The field of application of the invention is thus in the technical microbiology, the field of application of protein A, d. H. of the product produced according to the method lies on the one hand in biotechnology and on the other hand in human medicine (therapeutic, diagnostic).

Characteristic of the known state of the art

Microbial methods for producing protein A have been known since 1971 (see A. Arvidson et al., Acta Pathol, Microbiol., Scand., 79B, 1971, 399). As native protein A Bildnsr this Staphylococcus aureus strains were used, and first such producers in which the product is almost completely zellwandgundund. However, S. aureus parent material, which secretes the protein A into the culture medium, was also used very soon for the biosynthesis (A. Forsgren et al., J. Bacteriol., 107, 1971, 245, G. Masuda et al., Infect Immun., 2, 1975, 245).

With the work of S. Löfdahl, M. Uhlen et al., Proc. Natl. Acad. Be. USA, 80, 1983, 697, a method was first disclosed in which recombinant microorganism strains, in particular E. coli strains, were used for the microwave production of protein A. In the meantime, however, other process solutions for the production of this substance using recombinant bacterial strains, referring to Bacillus subtill strains, have also been reported (S.R. Fahnenstock & K.E. Fisher, Appl.Envir.Microbiol., 53, 1987, 379).

In all previous methods for protein A protein preparation, only affinite-chromatographic methods are used in the work-up or purification section, the target substance being bound to gamma-globulin, each fixed to a solid support, in the neutral range and subsequently reduced to values of about 3 by acidity reduction. 0 is eluted (fine purification by means of affinity chromatography on immobilized gamma-globulin).

These processing solutions are comparatively uneffective since the carriers used in them only undergo a special modification, i. H. can be used after an activation step, continue to have only a limited life, difficult to sterilize and are destructible by bacterial contaminants. In addition, their binding potency is not very high (1 milligram of gamma-globulin theoretically can bind only to O, 3 milligrams of protein; after immobilization of the globulin, often only a fraction of this theoretical capacity can be used), and with repeated use the carrier is a persistent wasting register their binding capacity. Furthermore, it is finally necessary in these methods to subsequently liberate the protein A obtained from the cleavage products by gel chromatography.

Object of the invention

The object of the invention is to provide highly purified protein A for the various uses mentioned.

Explanation of the essence of the invention The Ftlindung the task is based on a technical-microbiological process to rewrite, with the help of the Kuituifiltratender used for fermentation microorganisms in high yield highly purified protein A.

can be.

Eifino'uncjsgemäß this task is solved in its basic feature as follows: A piotein A-forming microorganism, i. either a Stamrr of the species Staphylococcus aureus or a meanwhile

available, for the biosynthesis of this protein capable recombinant microorganism (preferably a recombinant

BaKterienstamm), is submerged in a nutrient medium containing a carbon and a Slicksloffquelle and mineral salts

contains, for the duration of 8 hours to 30 hours at temperatures of 25 ⁰ C to 40 ° C and pH values in the range between

pH 6.0 and pH 8.0. Preferably, a dialysate nutrient medium is used for the fermentation batch. The biomass obtained is separated from the culture solution after completion of the fermentation, optionally following cell disruption. The resulting protein A-containing culture Übersland (the recovered protein A-containing Kulturfiltrai) is then treated with a wide-pore silica gel with pore sizes in the range of 10nm to 50nm. As a silicate adsorbent of this nature, a far-pored silica gel of the above specificity is preferred according to the method

from variety classification B1 or from the variety classification Si 100 or from the variety classification Si 300 or from the variety classification XWP500

used. This process step is based on the finding that silicate adsorbents of the above specificity, ie wide-pore silica gels, are capable of completely binding protein A from culture supernatants of microorganisms. In the following process step, the above-loaded wide-pore silica gel above specificity of the protein A, if necessary, after having been eliminated in a wash in an alkaline medium foreign proteins, with a solution of a monohydric or polyhydric alcohol or a ketone or an amine or an amide detached; and from the eluates obtained in this case, the protein A is finally isolated by means of ion exchange chromatography in a highly pure form. In its further embodiment, the invention is further characterized in that the far-pored silica gel of the variety classification chosen in each case in an amount of 0.2kg to 2.0kg per 1001 in the resulting after fermentation and biomass separation protein A-containing culture supernatant is stirred. If a wash cycle is subsequently required in the process, an ethanolic solution with soda additive, preferably a solution of 10 ml to 15 ml of ethanol per 100 ml of water with 1% soda, is used for this at pH values from pH 8.0 to pH 9.5. Additive, used. For the subsequent detachment of the target substance from the loaded wide-pore silica gel is now either

- At pH 10.5 an alkaline solution of ethylene glycol, optionally with the addition of glycine, of tris (hydroxymethyl) aminomethane, of triethylamine or of Polyoxyethylsorbitanmonolaurat, or

- At pH 2.0, an acidic ethylene glycol solution, optionally with one of the aforementioned additives, or

- At pH 2.0, an acidic ethanol solution, optionally with one of the aforementioned additives, or

- At pH 2.0, an acidic acetone solution, optionally with one of the above-mentioned additives, or

- At pH 10.0 Ms pH 11.0 an ethanolamine solution, optionally with one of the aforementioned additives, or

- At pH 10.5 a formamide solution, optionally with one of the aforementioned additives, or

- At pH 2.0 to pH 10.5, a urea solution, optionally with one of the aforementioned additives, used as eluent.

In the following step of the Gesarmverfahrens is now the eluent including dor recorded by him Impurities - optionally after an intermediate dialysis - separated by ion exchange chromatography and the

bound protein A finally eluted from the ion exchanger and - optionally after a re-dialysis-lyophilized. For separation of the eluent and of any impurities present (pigments,

Foreign proteins, etc.), a cation or an anion exchanger is optionally used here. In the preferred embodiment of the invention, in which in the microbiological pitting stage in a yeast dialysate Peptone nutrient medium, the Staphylococcus aureus strain Cowan l / M, i. a mutant containing the synthesized protein A in the Liberates culture solution, uses it for submerged fermentation, becomes in the introductory processing steps that from the collection

Of the above-mentioned pie classifications each used far-pored silica gel in an amount of 0.2kg to 2.0kg per 1001 in the protein A-containing culture supernatants stirred. After completion of the stirring, the respective silicate adsorbent used above quantitatively sedimentes the above specificity within a few minutes. For its washing, a solution of 10ml to 15ml of ethanol per 100ml of water with 1% soda additive is used. The silica gel recovered after the elution step can be used at least 15 times for the process of the adsorption step as indicated by appropriate control experiments.

The Ruindarstollung of the product in the final step of Gosamtverfahrens by cation exchange chromatography

is carried out, so as to separate both the respective eluent (selected from the above-mentioned

Total amount of such agents) as well as any impurities present as an exchanger preferably one CM-cellulose used; diose is used at pH values below pH 5.0. If the separation of the eluent and any impurities present, however, via an anion exchanger

made, so preferably a DEAE-cellulose is used; dioso is used at pH values above pH 5.5.

In this way white, impurity-free protein A is obtained, which in SDS electrophoresis is a band for

shows a relative molecular weight of about 50,000.

In the individual steps of the process, the protein Α-concentration was jowoils while using Mancini technique under Use of a 0.5% Sciiwemenormalserum containing agar determined. In connection with the application of the invention, the following additional advantages are also seen:

- To bind the protein A au; Microorganism culture supernatants of the kind set forth employ an adsorption material which is extremely inexpensive to prepare.

- The wide-pore silica gels used for adsorption are easily sterilizable; they are not attacked by bacteria and iodine sedimented from them, if it is added as by stirring in a protein A-containing culture supernatant, within a few minutes completely.

- Mild method described protein A-containing culture brothels of all kinds-whether cloudy, discolored or contaminated with bacteria - and in volumes of all sizes without additional effort.

The protein A obtained according to the invention is poor in cleavage fragments, so that the post-gelation of a gel filtration is not necessary.

-4- 286 721 Export description

example 1

Forty liters of a culture of Staphylococcus aurdus Cowan l / M obtained after fermentation in yeast dialysate-peptone medium are heated for 15 minutes at 80 ° C. Then the bacterial biomass in the separator is separated into culture supernatant - in this example case it contained 89 mg / l Protein A - are added at room temperature (dry weight) and stirred for 30 minutes to 60 minutes in a dry, wide-pored Kie algel of variety classification B1 (wet weight 1550 g.) After the stirrer has been switched off, the loaded kieselgol B1 is about 5 The supernatant is discarded, the crude silica gel is washed extensively with water and transferred to a β-1 vessel (for example a beaker or similar), followed in 2 portions with 2 liters of a 1% soda solution in each case Washed with 15% ethanolic water at pH 9.0 * ± 0.5 with stirring, the entire protein A remains bound but the majority of the foreign proteins are removed From this loaded silica gel 1 g of wet mass, to which 2.3 mg of protein A were bound, mt per 5 ml of the listed in Table 1 eluants ethanol, glycine, ethylene glycol, ethanolamine, formamide srwie urea mixed and after 2 hours of incubation in the in Tabe'le 1 extreme pH values dio Protein A-concentration in the supernatant determined (Mancini technique). The results are also shown in Table 1.

Example 2: To a liter of culture bursting with 36mg / l of protein A (to obtain this culture supernatant, the procedure is as in Example 1) are for a period of 60 minutes at room temperature 10g far-pored silica gel Grade classification 51300 (particle size 0.03mm) stirred. The loaded wide-pored silica gel is then

separated and then rinsed with 500 ml of water and with 250 ml of carbonate precipitate (1% sodium carbonate solution in 15% ethanol-containing water, pH 0.0 ± 0.5). Elution e: follows in the manner of Example 1 with 100 ml of a one molar solution of

Ethanolamine in water at pH 10.5, adjusted with NaOM. This gives 100 ml Eluit with 21.6 mg total protein A, which corresponds to a yield of 60%. Example 3: To a liter of culture supernatant with 36mg / l protein A (to obtain this culture supernatant, the procedure is as in Example 1) during a period of 60 minutes at room temperature 10g far-pored silica gel Grade classification Si 100 (particle size 0.03mm) stirred. The washing procedure is carried out as in Example 2 with water and

with carbonate buffer. For elution, which in turn takes place in the manner of Example 1, the charged loaded is far-pored

Silica gel with 100 ml of acid Actton-containing glycine buffer [0.1 M glycine in 50% aqueous acetone (v / v)] at pH 2.0

(set with KCI) offset.

One obtains 10% ORI with 21 mg total Pt'otein A; this corresponds to a yield of 58%.

BolspleU

10g wide-grained gravel organ of the variety classification XWP500 become in room temperature over 60 minutes in 1 liter

Culture supernatant stirred with .6mg / l protein. To obtain this culture supernatant, the procedure is as in Example 1. The separated loaded silica gel XWP500 is then treated according to Example 2 with water and carbonate buffer

washed and then eluted with 100 ml of 50% ethylene glycol water containing 0.1 M glycine (pH 10.5, adjusted with NaOH).

This gives 100 ml of eluate with 18.4 mg of protein A, which corresponds to a yield of 51%.

BeIpIeI 5

1 500 g of wet, washed, washed woit-pored silica gel of grade classification B1, to which 40 g of culture medium (30 mg / l) 1.2 g of Proteiri A are bound (for the recovery of this medium, for the adsorption and for the washing step, see Example 1), Waiden eluted with 5 liters of 0.05M glycine in 50% ethylene glycol wesser at pH 10.5 on a frit, and the

Pass is dissolved in Poitionon at 500ml each. The protein A was in the eluate 2 to 5 (2 liters Flüssigkeitsmongo). The protein A-haltigon eluates are preinominated, and the pH is adjusted to pH 4.4 with HCl. After standing over At night at 4 ° C, the supernatant is separated from the ousge'allonen precipitate. The protein A-holtige solution is then

over a column (cm diameter, 7 cm height) of CM-cellulose equilibrated with buffer A (0.1 M acetic acid with 2.7 ml of 25%

Ammonia per liter; pH 4.5; Conductivity 2,7mS / cm). After guards with buffer A to zero extinction (at 280nm)

is treated with Buffer B (0.1 M acetic acid with 7.5% w / w 25% ammonia, pH 5.6, conductivity 7.5 mS / cm). The elu & t will be against

Water diiilysiort and finally lyophilized. Yield: 600mg pure protein A

(corresponds to a relative yield of 50%).

Examples:

In 40 liters of Kulluf supernatant with 30my / l of protein A, obtained according to example 1, 500 g of dry, gypsum-soluble kiosolgol ore clarification B1 were introduced and the suspension was stirred for 60 minutes. The loaded wide-pore silica gel is washed and the protein A according to the instructions of Example 5 with 0.05 M glycine in ethylene glycol water Eluiort. For the final step, the protein A-containing eluates are combined and the acidity is adjusted to pH <t, 4 with HCl. The äzipitat to making ^one 'Pr abzontrifugiert; Thereafter, 200 ml of CM cellulose equilibrated with buffer A (0.0153 M citric acid plu3 0.022 M phosphate, pH 4.4) were stirred into the supernatant, and the cellulose was then spooled in a chromatography gel. After washing with Buffer A, Protein A is eluted from the CM cellulose using a linear gradient of 500 ml Buffer A and 500 ml Buffer B (0.0113Ni citric acid plus 0.029M phosphate, pH 5.6). Dio active fractions were combined and precipitated with ammonium sulfate at 70% saturation; the precipitate is dissolved in water and then dialyzed against water and finally against 0.01 M ammonium carbonate

 After lyophilization, a protic yield of 724 mg was achieved (corresponding to a relative yield of 60%).

Example 7

5 liters of a protein A-haltlgen ethylene glycol eluate, obtained using loaded wide-pore silica gel grade classification B1 of Example 5, with a total active ingredient content of 700mg protein A are after adjusting the pH to pH 7.5 above a DEAE Cellulose column (5 cm in diameter χ 7 cm in height) added with 0.05 M Tris-HCl; pH 7.5; equlllbriert had been. After rinsing with this buffer, elution is carried out with an addition of 0.5 M NaCl.

After dialysis of the eluate against water, a final yield of 520 mg protein A, calculated from the culture filtrate content, was obtained (corresponding to a relative yield of 74%).

Examples

Protein A-containing, washed wet mass silica gel of grade classification B1 (1 550 g) obtained according to Example 1 with 3.2 g of protein A is eluted in 3 portions of 1.5 liters of 50% ethanol solution in 0.1 M glycine -Buffer; pH 2.0; stirred (stirring time 30 minutes) and decanted after settling. The eluates are combined and the pH is adjusted to pH 4.4 with HCl.

After filtering the cloudy precipitate into the supernatant for the final step of the process 400 ml CM-Celluloso equilibrated with 0.1 M ammonium acetate; pH 4.4; stirred. The loaded cellulose is then rinsed on a frit and washed with the equilibration buffer. Re-elution is done with 2 liters of 0.1 M solution of the same buffer; pH 5.6; containing 1.0 M NaCl, and from the eluate, the protein A is precipitated with ammonium sulfate at 70% saturation, dialyzed and finally lyophylosed.

Yield: 1.9 g protein A

(corresponds to a relative yield of 60%).

Example 9

1500 g of wet mass of protein A-containing silica gel grade classification B1, obtained according to Example 1, to which from 40 liters of culture filtrate 3.6 g protein A were bound, in the elution step with 3 portions of 1.5 liters of 50% acetone solution containing 0 , 1 M glycine; pH 2.0; stirred for 30 minutes, and the eluates are then combined. After adjusting the pH to pH 4.4 and 2'entrifugation of the cloudy precipitate, the further purification of CM-cellulose according to Example 8.

Yield: 2.5 g protein A

(corresponds to a relative yield of 69%).

Example 10

Damp loaded far-pored silica gel grade classification B1, obtained in an amount of 100g according to Example 1, to which 0.29g protein A are bound in the elution step with 3 portions (of 100ml) 50% formamide is stirred, and the supernatants are united. The eluate is challenged with 0.1 M ammonium acetate buffer; pH 4.4; clialysiort and finely purified as in Example 8 to CM-cellulose.

Yield: 250 mg protein A

(corresponds to a relative yield of 86%).

Example 11

Wet bolsdenos wide-pore silica gel of variety classification B1, obtained in an amount of 100 g according to example 1, to which 0.29 g protein A are bound, in the elution step with 3 portions (of 100ml each) of 0.5M ethanolamine; pH 10.5; stirred, and the supernatants obtained are combined. After dialysis against 0.1 M ammonium acetate buffer; pH 4.4; The Foinreinigung again takes place on CM-cellulose as in Example 8

 Yield: 230 mg protein A

(corresponds to a relative yield of 80%).

Example 12

Wet Beladones far-pored silica gel grade classification B1, obtained in an amount of 100g according to Example 1, to which again 0.29g protein A gebundon are in the elution step with 3 portions (of 100ml) 0.1 M glycine buffer in 50% ethylene glycol ; pH 2.0; stirred, and the eluates are combined. The pH of the total eluate is adjusted to pH 4.4 with 4 N NaOH; then this solution is finely purified according to the procedure of Example 8 to CM-cellulose. Yield: 177mg Protein A

(corresponds to a relative yield of 61%).

Example 13

Wet Bolads wide-pore KioselgeldorSoitenklassifizierung B1, obtained in an amount of 100g according to Example 1, to which also 0.29g Protein A are bound, is in Elutionsjchritt with 3 servings (from jo 100ml) 0.1 M glycine buffer; pH?, 0; containing 6 M urea, stirred and the supernatants are combined. This eluate was first added to dialysis Wassei and then Ammüniumsulfat to 50% saturation. The precipitated protein A is again diclyzed against 0.02M Ammoniumbikarbonei sowio finally lyophilized. Yield: 205 mg protein A

(corresponds to a relative Ausboute of 71%).

Table 1 Eluted protein A as a function of the eluent used and of the pH for 1 g of woitporiges silica gel

(Variety classification B1, wet mass)

(1 g of silica gel was incubated with 5 ml of eluent for 2 hours at room temperature and the eluted protein A im

Supernatant determined.)

eluent _ψ Ethanol 30% -0.1 M glycine pH mg Ethanol 30% -0.1 M glycine Protein A Acetone 50% -0.1 M glycine imEluat Acetone 30% -0.1 mgIycin 9.5 0 Ethylene glycol 50% -0.1 M glycine 2.0 1.35 Ethylene glycol 50% -0.1 mgIycin 2.0 2.7 Ethylene glycol 50% ~ 0.05 M TRIS 2.0 2.0 Formamide 50% 10.5 1.5 Ethanolamine 0.5 M 2.0 1.6 Ethanolamine 1.0M 9.5 0.9 Urea M 7.0 2.7 Tween20 (1%) - 0.1 mg mycine 10.7 2.0 11.0 2.0 7.0 1.7 10.5 0.6

Claims (13)

1. A method for obtaining protein A on microbial Wega by - in a nutrient medium welchos a carbon and a nitrogen source and mineral salts containing a for protein Α biosynthesis more capable strain of the species Staphylococcus aureus, or a recombinant microorganism with this capability submerged for a period of 8 hours to 30 hours at temperatures of 25 ⁰ C to 4O ⁰ C and pH values in the range of pH 6.0 to pH 8.0 cultured, - * after completion of the fermentation, optionally following a cell digestion, the biomass obtained and separated - isolated in the final step of the overall process from the then present eluates by ion exchange chromatography, the pure product is characterized in that a) the protein A-containing culture supernatant with a wide-pore silica gel with pore sizes in the range of 10nm to 50nm treated and b) the target product from the now loaded silica gel above specificity with a solution of a mono- or polyhydric alcohol or a cotone or an amine or an amide detached 2. The method according to claim 1, characterized in that the far-pored silica gel above specificity from the variety classification B1 or
from the grade classification Si 100 or
from the grade classification Si 300 or
from the variety classification XWP500
in an amount of 0.2 kg to 2.0 kg per 1001 is stirred into said culture supernatant. 3. The method according to claim 1 and 2, characterized in that the loaded silica gel of the above specificity is subjected to a washing process in the alkaline medium before the separation step. 4. The method according to claim 3, characterized in that in the washing process at pH values between pH 8.0 and pH 9.5 an ethanolic solution with soda additive is used. 5. The method according to claim 4, characterized in that in the washing process, a solution of 10ml to 15ml of ethanol per 100ml of water with 1% soda additive is used. 6. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein A is replaced from the loaded silica gel above specificity with alkaline ethylene glycol solution at pH 10.5. 7. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein A is replaced from the loaded silica gel above specificity with acidic ethylene glycol solution at pH 2.0. 8. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein A is replaced from the loaded silica gel above specificity with acidic ethanol solution at pH 2.0. 9. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein A is replaced from the loaded silica gel above specificity with acidic acetone solution at pH 2.0. 10. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein Avom loaded silica gel above specificity is replaced with ethanolamine solution in an alkaline medium. 11. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that protein A is replaced by the loaded silica gel of the above specificity with formamide solution at pH 10.5. 12. The method according to claim 1 or 2 and optionally 3 to 5, characterized in that o'aß protein A is replaced by laden silica gel above / specificity with urea solution at pH 2.0 to pH 10.5. 13. The method according to claim 6 to 12, characterized in that there at the replacement there? respective eluents with an additive of glycine or of tris (hydroxymethyl) aminomethane or of triethylamine or of polyoxyethyl sorbitan monolaurate
is used.