U.S. Patent: 5314993 - Process for preparing high purity protein A preparation

Back to EveryPatent.com

United States Patent	*5,314,993*
Love , et al.	May 24, 1994

Process for preparing high purity protein A preparation

Abstract

The invention concerns a process for purifying protein A preparations to high purity with high product yield. Where the protein A is obtained from a Gram-negative recombinant microbe hosting a vehicle containing a gene encoding protein A, the protein A is purified to high purity, and, advantageously, to very low levels of endotoxin. The protein A preparations made via the invention process are useful in therapeutic application, e.g., therapeutic plasma exchange, as well as for other well-known uses of protein A.

Inventors:	Love; Richard N. (Watertown, MA); Profy; Albert T. (Cambridge, MA)
Assignee:	Repligen Corporation (Cambridge, MA)
Appl. No.:	833584
Filed:	February 10, 1992

Intern'l Class: C07K 015/04; C07K 003/24; C07K 003/20; C07K 003/28

Field of Search: 530/350,416,419,412,418,427

References Cited U.S. Patent Documents

4412985	Nov., 1983	Shanbrom	424/78.
4673733	Jun., 1987	Chandra et al.	530/344.

Other References

Abstract, WPI Acc No. 77-42316y/24, Dialog File 350, (JP52054090), 1977.
Kayser, A. 1969, C. R. Soc. Biol. 63(3):643-648.
Chemical Abstract No. 130512r. Kayser, A. 1969, C. R. Soc. Biol. 163(3):643-648.
Sofer, G. 1984, Bio/Technology, Dec. pp. 1035-1038.
Chemical Abstract No. 106:137001c, Kudo et al. JP61,224,997.
Chemical Abstract No. 103:176670c. Sehi et al. 1985, Microbiol Immunol, 29(6):559-563.
Lofdahl et al. 1983, Proc. Natl. Acad. Sci. 80:697-701.
Pharmacia, 1986, Separation News vol. 13(6) pp. 2-8, Publication SN50-01-339.
Scoper, R. K. 1982, Protein Purification, Springer-Verlag, N.Y., pp. 52-101.
Schrezenmeier et al. 1987, J. Immunol. Meth. 105:133-137.

Primary Examiner: Furman; Keith C.
Attorney, Agent or Firm: Saliwanchik & Saliwanchik

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 459,577, filed Mar. 7, 1990, now abandoned; which is a division of application Ser. No. 083,544, filed Aug. 6, 1987, now abandoned, which is a continuation-in-part of copending application Ser. No. 030,934, filed Mar. 26, 1987, now abandoned.

Claims

We claim:

1. A process for purifying protein A from a protein A-containing solution or other protein A preparation which comprises

(a) heating a protein A-containing solution at a temperature of about 50.degree. C. to about 80.degree. C. for about 30 minutes, causing some contaminating protein to precipitate;

(b) loading an ion-exchange column by passing the protein A-containing solution over said column;

(c) washing the loaded column with a solution comprising a non-ionic detergent;

(d) eluting protein A from the ion-exchange column; and

(e) precipitating the eluted protein A with ethanol to give a substantially pure preparation of protein A.

2. A process, according to claim 1, wherein said protein A-containing solution or other protein A preparation results from a recombinant DNA system designed to express protein A.

3. A process, according to claim 2, wherein said recombinant DNA system comprises a prokaryotic host comprising an extrachromosomal element encoding protein A.

4. A process, according to claim 3, wherein said prokaryotic host is Escherichia coli.

5. A process, according to claim 4, wherein said Escherichia coli host is Escherichia coli NRRL B-15910.

6. A process, according to claim 1, wherein the protein A-containing solution is heated at about 80.degree. C. for about 30 minutes.

7. A process, according to claim 1, wherein said ion-exchange column comprises primary, secondary, tertiary, or quaternary amine functionalities.

8. A process, according to claim 7, wherein said functionalities are diethylaminoethyl support, aminoethyl support, or quaternary amino support, wherein said support is an insoluble hydrophilic substance.

9. A process, according to claim 8, wherein said insoluble hydrophilic substance is cellulose, agarose, or crosslinked dextran.

10. A process, according to claim 1, wherein the ion-exchange column is an anion- or cation-exchange column.

11. A process, according to claim 10, wherein the anion exchange column is diethylaminoethyl-agarose.

12. A process, according to claim 10, wherein the cation-exchange column is carboxymethyl-agarose.

13. A process, according to claim 11, wherein the anion-exchange column is washed with a biologically compatible buffer solution between about pH 6 and about pH 10.

14. A process, according to claim 12, wherein the cation-exchange column is washed with a biologically compatible buffer solution between about pH 3 and about pH 6.

15. A process, according to claim 13, wherein the buffer solution comprises 25 mM Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3, 2 mM potassium ethylenediaminetetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.2% polyoxyethyleneoctylphenol ether.

16. A process, according to claim 13, wherein the initial anion-exchange column wash is followed by a wash with 25 mM Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3.

17. A process, according to claim 10, wherein the anion-exchange column is eluted with a buffer solution consisting of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3 and 150 mM potassium chloride.

18. A process, according to claim 14, wherein the buffer solution comprises 0.5M sodium acetate pH 4.5, and 1% polyoxyethyleneoctylphenol ether.

19. A process, according to claim 14, wherein the initial cation-exchange column wash is followed by a wash with 50 mM sodium acetate, pH 4.5.

20. A process, according to claim 10, wherein the cation-exchange column is eluted with buffer solutions consisting of 50 mM sodium acetate, pH 4.5, and 0.1N potassium chloride followed by 50 mM sodium acetate, pH 4.5, and 0.3N potassium chloride.

21. A process for purifying protein A from a protein A-containing solution or other protein A preparation which comprises

(a) heating a protein A-containing solution at a temperature of about 50.degree. C. to about 80.degree. C. for about 30 minutes, causing some contaminating proteins to precipitate;

(b) precipitating the protein A with ethanol and re-dissolving the pellet in a buffer;

(c) loading an ion-exchange column by passing the re-dissolved protein A-containing solution over said column;

(d) washing the loaded column with a solution comprising a non-ionic detergent;

(e) eluting protein A from the ion-exchange column; and

(f) precipitating the eluted protein A with ethanol to give a substantially pure preparation of protein A.

22. A process, according to claim 21, wherein said protein A-containing solution or other protein A preparation results from a recombinant DNA system designed to express protein A.

23. A process, according to claim 22, wherein said recombinant DNA system comprises a prokaryotic host comprising an extrachromosomal element encoding protein A.

24. A process, according to claim 23, wherein said prokaryotic host is Escherichia coli.

25. A process, according to claim 24, wherein said Escherichia coli host is Escherichia coli NRRL B-15910.

26. A process, according to claim 21, wherein the protein A-containing solution is heated at about 80.degree. C. for about 30 minutes.

27. A process, according to claim 21, wherein said ion-exchange column comprises primary, secondary, tertiary, or quaternary amine functionalities.

28. A process, according to claim 27, wherein said functionalities are diethylaminoethyl support, aminoethyl support, or quaternary amino support, wherein said support is an insoluble hydrophilic substance.

29. A process, according to claim 28, wherein said insoluble hydrophilic substance is cellulose, agarose, or crosslinked dextran.

30. A process, according to claim 21, wherein the ion-exchange column is an anion- or cation-exchange column.

31. A process, according to claim 30, wherein the anion exchange column is DEAE-agarose.

32. A process, according to claim 30, wherein the cation-exchange column is carboxymethyl agarose.

33. A process, according to claim 31, wherein the anion-exchange column is washed with a biogically compatible buffer solution between about pH 6 and about pH 10.

34. A process, according to claim 32, wherein the cation-exchange column is washed with a biologically compatible buffer solution between about pH 3 and about pH 6.

35. A process, according to claim 33, wherein the buffer solution comprises 25 mM Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3, 2 mM potassium ethylenediaminetetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride, and 0.2% polyoxyethyleneoctylphenol ether.

36. A process, according to claim 33, wherein the initial anion-exchange column wash is followed by a wash with 25 mM, Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3.

37. A process, according to claim 30, wherein the anion-exchange column is eluted with a buffer solution consisting of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride, pH 8.3 and 150 mM potassium chloride.

38. A process, according to claim 34, wherein the buffer solution comprises 0.5M sodium acetate, pH 4.5, and 1% polyoxyethyleneoctylphenol ether.

39. A process, according to claim 34, wherein the initial cation-exchange column wash is followed by a wash with 50 mM sodium acetate, pH 4.5.

40. A process, according to claim 30, wherein the cation-exchange column is eluted with buffer solutions consisting of 50 mM sodium acetate, pH 4.5, and 0.1N potassium chloride followed by 50 mM sodium acetate, pH 4.5, and 0.3 N potassium chloride.

41. The process of claim 6, wherein said protein A-containing solution has a basic pH.

42. The process of claim 26 wherein said protein A-containing solution has a basic pH.

Description

BACKGROUND OF THE INVENTION

Downstream processing of expressed recombinant proteins is an important consideration in the utilization of cloned gene products. Specifically, purification at industrial levels must supply a protein of sufficient purity in a cost effective manner.

Protein A is a well-known, useful protein originally derived from the cell wall of Staphylococcus aureus. It has the ability to bind the Fc region of most mammalian antibodies of class IgG. Commercial applications of protein A utilizing this property include large-scale affinity purification of monoclonal antibodies, many of which will have therapeutic applications; therapeutic plasma exchange, in which the circulating immune complexes from blood plasma are removed via binding to immobilized protein A; as well as numerous laboratory applications based on immunochemical or histochemical procedures.

To meet these commercial demands, protein A must be economically produced in large quantities and, for therapeutic applications, the protein A must be greater than 99% pure as determined by a variety of assay methods including SDS polyacrylamide gel electrophoresis (PAGE) and size-exclusion HPLC. In addition, contaminating endotoxin levels, especially important in protein A preparations produced from Gram-negative hosts, must be very low, on the order of <1.0 endotoxin units (E.U.)/mg protein A as determined by the gel clot assay (LAL).

There are currently two published methods used to purify protein A. One method uses an IgG affinity column, and the other is a multistep, small scale process which includes the use of a diethylaminoethyl (DEAE) ion-exchange column followed by the use of a sizing column.

The first method utilizes the binding of protein A to the Fc region of the IgG molecule. As developed by Hjelm et al. (Hjelm, H., Hjelm, K., Sjoquist, J [1972] FEBS Letters 28:73-76), IgG is bound to an insoluble matrix which is then packed into a column. A protein A-containing solution is passed over the column at neutral pH in 0.1M H.sub.2 PO.sub.4.sup.-. Under these conditions protein A is selectively removed from the solution due to its interaction with the immobilized IgG. The protein A is eluted from the column with 0.1M glycine-HCl, pH 3.0 and comes off with the buffer front.

Protein A also has been purified using a multistep procedure (Sjoquist, J., Meloun, B., Hjelm, H., [1972] Eur. J. Biochem. 29:572-578). S. aureus, strain Cowan I (NCTC 8530, National Collection of Type Cultures, Central Public Health Laboratory, London, England) is pelleted and resuspended in 0.05M TRIS-HCl [TRIS=Tris(hydroxymethyl)aminomethane], 0.145M NaCl pH 7.5 and heated to 37.degree. C. Lysostaphin and DNAase are added and digestion allowed to go to completion as monitored by absorbance at 520 nm. After the absorbance values become constant, the suspension is centrifuged and the supernatant is treated with 5N HCl to lower its pH to 3.5. The resulting precipitate is removed by centrifugation and the supernatant adjusted to pH 7.0 with 5N NaOH. Ammonium sulfate is added to make an 80% saturated solution. This material is centrifuged and the precipitate removed, redissolved in 0.1M ammonium bicarbonate, pH 8.0, and applied to a DEAE-SEPHADEX (SEPHADEX is a tradename of Pharmacia Inc., Piscataway, N.J.) column equilibrated in 0.1M ammonium bicarbonate, pH 8.0. Under these conditions, the protein A molecule will bind to the positively charged DEAE groups on the column. Protein A is then eluted by increasing the ionic strength of the column buffer via a linear gradient to 0.4M ammonium bicarbonate, pH 8.0.

The protein A removed from the column is concentrated and applied to a SEPHADEX G-100 column equilibrated in 0.05M Tris.HCl, 1.0M NaCl pH 8.0. On such a column proteins are separated according to size--larger molecules are excluded from the interior of the porous SEPHADEX.RTM. and therefore pass through the column more rapidly than smaller molecules which can enter the column packing.

While these purification schemes give satisfactory results for laboratory use, they are inadequate for industrial-scale applications. The problems associated with the first procedure include the high cost of preparation and maintaining an immobilized IgG column. The IgG must be isolated before any work toward purifying protein A can begin. Also, the immobilization of IgG may involve the use of a highly toxic material, cyanogen bromide. Another problem occurs because a biological molecule is part of the column packing. This means that much care must be taken to prevent its contamination or degradation and concomitant loss of binding activity. In spite of such measures, column life is considerably less than that of other properly maintained column packings.

The second purification scheme, discussed above, also is limited for large-scale work due to several reasons. This is a multistep procedure involving acid precipitation, ammonium sulfate precipitation, and buffer exchange before reaching the ion-exchange and sizing column steps. Because each step of a purification results in some loss of material, the overall number of steps should be kept to a minimum. Another drawback to this method is the amount of ammonium bicarbonate used during the ion-exchange chromatography. The cost of using a gradient from 100 mM to 400 mM ammonium bicarbonate would be a substantial portion of the purification cost. The most serious drawback of the procedure, however, is the use of a sizing column. While such columns are suitable for laboratory operations, use of a sizing column at industrial levels is undesirable because capacity is very low (sample size is limited to 2-5% of the column volume), processing time is quite slow compared to other chromatographic methods, and, finally, sizing columns are often troublesome sources of contaminating endotoxins due to bacterial growth in the column.

Finally, both procedures, discussed above, involve the use of pH 3.0-3.5 solutions. This is not suitable for use in the purification of recombinant protein A expressed in E. coli as these organisms contain proteases which will degrade protein A at low pH's.

Thus, there is a need in the art for an efficient process to purify protein A-containing solutions on an industrial scale to obtain high-purity protein A preparations with acceptable low levels of endotoxin. The need is particularly great in the recombinant DNA art of producing protein A, as shown above.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns an efficient large-scale process for purifying a protein A-containing solution or other protein A preparation. The final yield, advantageously, is extremely high and purity of the final product is greater than 99%. In addition, the final product is suitable for therapeutic applications because of its extremely low endotoxin levels, <1.0 E.U./mg protein A. More specifically, the subject invention process utilizes a heat step in which the majority of the contaminating proteins are precipitated.

The resulting clarified protein A-containing solution is applied to an ion-exchange material which is packed into a column. This material can be an anion-exchange or a cation-exchange matrix. The anion-exchange matrix can consist of primary, secondary, or quaternary amino groups, e.g., aminoethyl, diethylaminoethyl, and diethyl(2-hydroxypropyl)aminoethyl groups, bound to a solid support. The cation-exchange matrix can consist of a negatively charged group, such as carboxymethyl or sulfopropyl groups, bound to a solid support. The solid support may be any insoluble, hydrophilic substance, e.g. cellulose, agarose, TRISACRYL (Reg. Trademark, Reactifs IBF-Societe Chimique Pointet-Girard, Villeneuve-La-Garenne, France), or crosslinked dextran. Anion-exchange materials are available under the trade names DEAE-SEPHAROSE FAST FLOW, Q SEPHAROSE FAST FLOW, and DEAE-SEPHACEL (Reg. Trademarks, Pharmacia, Inc., Piscataway, N.J.). Cation-exchange materials are available under the trade names CM-SEPHAROSE FAST FLOW, S SEPHAROSE FAST FLOW, and CM-SEPHACEL (Pharmacia).

Upon washing with a buffer containing a non-ionic detergent followed by the application of an elution buffer with increased ionic strength or changed pH, this column yields a protein A solution which is typically in excess of 95% purity, with endotoxin levels attainable to below 1.0 E.U./mg protein A. The protein A is then precipitated by the addition of ethanol. Contaminating proteins are removed with the supernatant.

DETAILED DISCLOSURE OF THE INVENTION

The invention process comprises:

1. heating a protein A-containing solution at a temperature of about 50.degree. C. to about 100.degree. C. for about 10 min to about 60 min.;

2. passing the redissolved protein A-containing solution over an ion-exchange column;

3. eluting protein A from the ion-exchange column; and

4. precipitating the eluted protein A with ethanol to give a substantially pure preparation of protein A.

Optionally, a precipitation step can be added after step 1. In this step the protein A can be precipitated with ethanol. The resulting protein A pellet can be dissolved in a buffer.

A washing step can be used, advantageously, after step 3 above, to remove contaminating proteins which are not removed in the heating step. As disclosed above, the majority of contaminating proteins are removed by the heating step. Thus, the heating step is critical.

The range of temperature and time for the heating step can be varied to give the best balance of purity and yield of protein A. This adjustment is well within the skill of a person in the art and does not require undue experimentation.

Where the starting protein A-containing solution is obtained from a recombinant source, e.g., a protein A E. coli culture, then the protein A preparation obtained by the invention process, advantageously, has a low level of endotoxin.

Since the greatest utility for the subject invention process will be in recombinant protein A recovery systems, the invention process is specifically exemplified hereinafter by reference to such a system.

Several features of this process make it quite suitable for industrial-scale production of therapeutic-quality protein A. Process time is kept to a minimum as each step in the process is a batch step with corresponding advantages in throughput. Another advantage of the batch nature of each of the steps is their large capacity-each is limited primarily by the volume of the containers or columns employed. The high throughput and large capacity of this process is in sharp contrast with the limitations of any process utilizing sizing columns. In addition, the column matrix used in the ion-exchange portion of this process is very stable, resulting in low maintenance costs as well as limiting the number of times it must be replaced. This avoids those problems typically associated with the use of bound IgG in affinity chromatography.

The reduction in endotoxin levels to the very low levels necessary for therapeutic applications is possible because of the buffer composition used in the ion-exchange column. The use of a non-ionic detergent allows the disassociation of protein A and endotoxin resulting in a reduction to less than 1.0 E.U./mg protein A, as opposed to the >10 E.U./mg protein A levels which are obtained when no detergent is used in the process. An affinity chromatography column can be used in place of the ion-exchange column.

Finally, the use of ethanol precipitation in this process combines purification, buffer exchange, and concentration in a single step. Such efficiency is critical to industrial scale processes.

The Materials and Methods used in this disclosure of the invention process are as follows:

(1) Preparation of Protein A via Recombinant Host Microbe

Recombinant host E. coli NRRL B-15910 was grown under suitable conditions to prepare protein A. Fermentation was performed in a 500-L ABEC fermenter (ABEC, Inc. Bethlehem, Pa.) fitted with dO.sub.2 and pH control. Recombinant cells were grown at dO.sub.2 of greater than 50% (air=100%) of saturation at the pH indicated. pH was adjusted by the addition of 85% lactic acid (U.S. Biochemical, Cleveland, Ohio). Foam was controlled by the addition of antifoam B (Sigma Chemical, St. Louis, Mo.). Fermentation temperature was 34.degree. C. All fermentations were conducted with a final volume of 350 L in modified 2% medium.

Modified 2% Medium

2.0 g/L potassium phosphate, monobasic (Mallinckrodt Inc., St. Louis, Mo.)

2.0 g/L potassium phosphate, dibasic (Mallinckrodt Inc.)

2.0 g/L sodium phosphate, dibasic, heptahydrate (Mallinckrodt Inc.)

20.0 g/L casein peptone (Marcor Development Corp., Hackensack, N.J.)

20.0 g/L Casamino acids (Marcor Development Corp.)

20.0 g/L yeast extract (Marcor Development Corp.)

20 mg/L chloramphenicol (U.S. Biochemical)

pH adjusted to 6.8 with 0.1N NaOH

E. coli NRRL B-15910 was deposited on Nov. 20, 1984. It is available to the public for experimental use only upon the grant of a patent which discloses this accession number. It should be understood that the availability of this deposit does not constitute a license to practice the subject invention in derogation of patent rights granted for the subject invention by governmental action. The culture deposit is in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL) Northern Regional Research Center, 1815 N. University Street, Peoria, Ill. 61604 USA.

(2) Buffers for Anion-Exchange Chromatography

Any biologically compatible buffer between about pH 6 and about pH 10 can be used in the subject invention. These buffers may or may not contain substances suitable for inhibiting proteases or stabilizing protein products against oxidation. In addition, the presence of a non-ionic detergent is necessary in the specified column equilibration and wash buffers. Below are buffers which can be used.

    ______________________________________
    Anion-Exchange Column
                    0.25M TRIS-HCl pH 8.3
    Equilibration Buffer #1*
                    2 mM potassium ethylenediamine-
                    tetraacetic acid (KEDTA)
                    0.1 mM phenylmethylsulfonyl-
                    fluoride (PMSF)
                    0.2% TRITON X-100
    Anion-Exchange Column
                    0.25M TRIS-HCL pH 8.3
    Equilibration Buffer #2*
                    2 mM KEDTA
                    0.1 mM PMSF
                    0.2% TRITON X-100
    Anion-Exchange Column
                    Same as Anion-Exchange
    Wash Buffer #1* Column Equilibration Buffer #2
    Anion-Exchange Column
                    25 mM TRIS-HCl pH 8.3
    Wash Buffer #2*
    Anion-Exchange Column
                    25 mM TRIS-HCl pH 8.3
    Elution Buffer* 150 mM KCl
    ______________________________________

(3) Buffers for Cation-Exchange Chromatography

Any biologically compatible buffer between about pH 3 and pH 6 can be used for the subject invention These buffers may or may not contain substances suitable for inhibiting proteases or stabilizing protein products against oxidation. In addition, the presence of a nonionic detergent is necessary in the specified column equilibration and wash buffers. Below are buffers which can be used.

    ______________________________________
    Cation-Exchange Column
                    0.5M sodium acetate (pH 4.5)
    Equilibration Buffer #1*
                    1% TRITON X-100
    Cation-Exchange Column
                    50 mM sodium acetate (pH 4.5)
    Equilibration Buffer #2*
                    1% TRITON X-100
    Cation-Exchange Column
                    Same as Cation-Exchange
    Wash Buffer #1* Column Equilibration Buffer #2
    Cation-Exchange Column
                    50 mM sodium acetate (pH 4.5)
    Wash Buffer #2*
    Cation-Exchange Column
                    50 mM sodium acetate (pH 4.5)
    Elution Buffer #1*
                    0.1N KCl
    Cation-Exchange Column
                    50 mM sodium acetate (pH 4.5)
    Elution Buffer #2*
                    0.3N KCl
    ______________________________________
     *Buffers are prepared pyrogenfree and are filtered through a 0.20 nm
     depyrogenated filter prior to use.

(4) Analytical Size-Exclusion HPLC

A 7.5.times.300 mm LKB TSK G3000 SW column (LKB) is equilibrated in 100 mM potassium phosphate pH 6.8. A chart speed of 0.5 cm/min is used with a flow rate of 0.5 ml/min. A 15-.mu.g peak will be 80% full scale at 2.0 AUFS (Absorption Units Full Scale, 214 nm).

(5) Gel Electrophoresis

A 1 mg/ml solution of protein A is made by the addition of 1 ml of the 10 mg/ml test solution to 9 ml of water. 100 .mu.l of this solution is added to 100 .mu.l of a solution containing 50% glycerol, 5% sodium dodecyl sulfate, 5% 2-mercaptoethanol and a trace of bromphenol blue. This mixture is heated to 100.degree. C. for 10 min, then cooled to room temperature. 10 .mu.l of this solution (5 .mu.g protein A) are electrophoresced on a 12% acrylamide SDS gel prepared according to the method of Laemmli (laemmli, U.K. [1970] Nature 227: 680-685). The separating gel contains 9.6 ml of acrylamide solution (37.5% acrylamide, 1% bisacrylamide), 7.5 ml of 1.5M Tris-HCl pH 8.3 buffer, 0.15 ml of 20% SDS, 9.6 ml of degased water, 0.75 ml of 1.5% ammonium persulfate, and 2.4 ml of 0.5 tetramethylethylenediamine (TEMED). The stacking gel contains 0.96 ml acrylamide solution, 2.5 ml of 0.5M TRIS-HCl pH 6.8, 0.05 ml 20% SDS, 5.0 ml of degased water, 0.7 ml 1.5% ammonium persulfate, and 0.8 ml of 0.5% TEMED.

(6) Limulus Amebocyte Lysate (LAL) Assay

Endotoxin levels of purified protein A were determined using the procedure recommended by the manufacturer (Associates of Cape Cod, Inc., Woods Hole, Mass.).

(7) Sterility

The absence of microbial contamination in the purified protein A was demonstrated by plating 100 .mu.l of a 10 mg/ml test solution of purified protein A on blood agar and YT plates (8 g tryptone, 5 g yeast extract, and 5 g NaCl/L). The plates were incubated for 48 hr at 37.degree. C.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1--Lysis

A 12-kg wcw (wet cell weight) cell pellet of E. coli NRRL B-15910, containing a plasmid coding for the production of protein A, is resuspended in homogenization buffer (e.g., 8.75 mM TRIS-HCl, pH 8.3, containing 2 mM KEDTA and 0.6M PMSF). The cell suspension is passed through a DYNO-MILL KDL-15 Model (DYNO-MILL [a bead mill] supplied by Impandex, Maywood, N.Y.). The DYNO-MILL is charged with 10 L of dry glass beads (0.5-0.7 mm diameter) and operated at the highest speed setting. The cell suspension is fed to the DYNO-MILL at a rate of 1200 ml/min. After the cell suspension has passed through the bead mill, the mill is washed with an additional 20 L of homogenization buffer.

The lysate is clarified by passage through a 0.20-.mu.m Microgon filter (Microgon, Laguna Hills, Calif.). The retained volume is taken to 40% of the starting volume and washed with 3 volumes of additional homogenization buffer. The pH of the filtrate is adjusted to about 8.3 with 1N NaOH. The pH range can be about 8.2 to about 8.4.

Example 2--Heat Step

The pH 8.3 clarified lysate, obtained in Example 1, is pumped into a jacketed steam-heated vessel (ABEC) and heated to about 80.degree. C. for about 30 min. The resulting precipitate is allowed to settle and the supernatant decanted. The precipitate contains only contaminating proteins and is discarded.

The protein A-containing supernatant is clarified by passage through a 0.2 .mu.m Microgon filter as described in Example 1. The pH of the filtrate is adjusted to about 8.3 with 1N NaOH and the conductivity adjusted to 4.5 mS/cm by diluting with distilled water.

Example 3--First Ethanol Precipitation Step (Optional)

The filtrate from the heat precipitation step is passed through a 10,000 NMWL spiral filter, e.g., Amicon S 10Y30, Amicon Corp., Danvers, Ma, until the retentate volume is reduced to 10 L. Ethanol is added to the retentate to a final concentration of 70%, and the protein A is allowed to precipitate. The supernatant is decanted and the pellet resuspended in 100 L of a suitable buffer (e.g., 25 mM Tris-HCl, pH 8.3, containing 2 mM KEDTA and 0.1 mM PMSF).

Example 4--Anion--Exchange Column

The resuspended pellet, obtained in Example 3, is loaded onto a 63.times.15 cm column packed with DEAE-SEPHAROSE FAST FLOW (Pharmacia). A 0.20-.mu.m filter is connected to the inlet port of this column to prevent microbial contamination. Prior to loading, the packed, depyrogenated column (depyrogenated according to packing manufacturer's instructions) is equilibrated by pumping 200 L of Anion-Exchange Column Equilibration Buffer #1 through the column at a flow rate of 8 L/min. (This flow rate is maintained through all ion-exchange column operations.) 200 L of Anion-Exchange Column Equilibration Buffer #2 are then pumped through the column. The column is then loaded with the pH 8.3 filtrate. The column is washed with 400 L of Anion-Exchange Column Wash Buffer #1, followed by 400 L of Anion-Exchange Column Wash Buffer #2. Finally, the column is eluted with 100 L of Anion-Exchange Column Elution Buffer. The eluted peak is collected and assayed for protein A using size-exclusion HPLC and for endotoxin using the LAL assay. It is found that most of the contaminating proteins remaining after the heat step are removed by the column wash step. In addition, a 10-fold decrease in endotoxin levels is routinely obtained. This step may be repeated to obtain endotoxin levels as low as necessary.

Example 5--Cation-Exchange Column

The pH of the resuspended ethanol pellet from Example 3 is adjusted to 4.5 and the conductivity to 3.5. It is then loaded on a 63.times.15 cm column packed with CM-SEPHAROSE FAST FLOW. A 0.20-.mu.m filter is connected to the inlet port of this column to prevent microbial contamination. Prior to loading, the packed, depyrogenated column (depyrogenated according to the manufacturer's instructions) is equilibrated by pumping 200 L of Cation-Exchange Column Equilibration Buffer #1 through the column at a flow rate of 8 L/min. (This flow rate is maintained through all column operations.) 200 L of Cation-Exchange Column Equilibration Buffer #2 are then pumped through the column. The column is then loaded with the resuspended ethanol pellet. The column is next washed with 400 L of Cation-Exchange Column Wash Buffer #1 and then with 400 L of Cation-Exchange Column Wash Buffer #2. Next, the column is washed with 100 L of Cation-Exchange Column Elution Buffer #1. Finally, the column is eluted with Cation-Exchange Column Elution Buffer #2. The eluted peak is collected, neutralized, and assayed for protein A using size-exclusion HPLC and for endotoxin using the LAL assay. Final endotoxin levels are routinely less than 1 E.U./mg.

Example 6--Ethanol Precipitation

The eluted protein A from Examples 4 or 5, at the desired endotoxin level, is then precipitated by dilution with 3 parts ethanol to 1 part eluate. The protein A precipitate is allowed to settle and the ethanol supernatant is discarded. The 99% pure protein A pellet is resuspended in the buffer of choice.

Example 7--Reducing Endotoxin Levels in Other Protein Preparations

The above examples use a protein A preparation as the starting material. Endotoxin levels in other protein preparations also can be reduced by following essentially the same procedure, to wit:

(a) binding of the protein in the protein preparation to an immobilized support,

(b) washing the immobilized support containing the bound protein with a non-ionic detergent, and

(c) eluting the protein from the immobilized support with a pyrogen-free biologically compatible buffer solution to obtain a protein preparation with a low level of endotoxin.

Any protein preparation can be processed in the above manner.

The removal of contaminating proteins from a protein A-containing solution by heating said solution to a temperature of about 50.degree. C. to about 100.degree. C. for about 10 min to about 60 min is effective when the protein A is derived from either a recombinant organism or from Staphylococcus aureus.

Top

Intern'l Class:	C07K 015/04; C07K 003/24; C07K 003/20; C07K 003/28
Field of Search:	530/350,416,419,412,418,427