METHOD FOR PURIFYING IMMUNOGLOBULIN SOLUTIONS

Abstract

Herein is reported a method for purifying cell cultivation supernatants either directly after fermentation or after one or more preliminary purification steps, such as protein A affinity chromatography. By adjusting the pH value in the acid range and subsequent incubation of the acidified solution host cell nucleic acid and host cell protein can be precipitated but the target polypeptide remains in solution. Thereafter the precipitate and therewith the contaminating host cell components can be removed by a simple physical separation step.

Claims

1. A method for producing an immunoglobulin comprising the following steps: i) adding a solution consisting of an acid and water to a cell cultivation supernatant from which cells and cell debris have been removed for adjusting the pH value to a value of from pH 4.5 to pH 5.5, whereby the solution is essentially free of divalent cations, ii) incubating the pH adjusted cell cultivation supernatant, and iii) removing the precipitate from the incubated cell cultivation supernatant and thereby producing the immunoglobulin, wherein the cell cultivation supernatant comprises the immunoglobulin at a concentration of not more than 10 mg/ml, wherein the concentration of the added acid is 5.5 mol/1 or lower.

2. The method according to claim 1, characterized in that at least 90% of the immunoglobulin remains in solution during the incubating step.

3. The method according to claim 1, characterized in that the incubating is at a temperature of from 2° C. to 10° C.

4. The method according to claim 3, characterized in that the incubating of the pH adjusted cell cultivation supernatant is at a temperature of about 4° C.

5. The method according to claim 1, characterized in that the incubating the pH adjusted supernatant is for about at least 2 hours.

6. The method according to claim 5, characterized in that the incubating is for about 2 hours to about 72 hours.

7. The method according to claim 6, characterized in that the incubating is for about 2 hours to about 48 hours.

8. The method according to claim 7, characterized in that the incubating is for about 24 hours.

9. The method according to claim 1, characterized in that the immunoglobulin is of subclass IgG1 and the incubating is for about 2 hours to about 48 hours at a pH value of from pH 4.5 to pH 3.5.

10. The method according to claim 1, characterized in that the immunoglobulin is of subclass IgG4 and the incubating is for about 2 hours to about 30 hours at a pH value of from pH 5.5 to pH 4.5.

11. The method according to claim 1, characterized in that the removing is by a method selected from sedimentation, decantation, filtration, settlement, and centrifugation.

12. The method according to claim 1, characterized in that the acid is selected from acetic acid, citric acid, hydrochloric acid, and phosphoric acid.

13. The method according to claim 1, characterized in that the concentration of the acid is of from 1.5 mol/1 to 5.5 mol/1.

14. The method according to claim 12, characterized in that the acid is acetic acid with a concentration of from 1.5 mol/1 to 4 mol/1.

15. The method according to claim 1, characterized in that the concentration of the immunoglobulin is of from 1 mg/ml to 5 mg/ml.

16. The method according to claim 1, characterized in that the cell is a CHO cell.

17. The method according to claim 1, characterized in that the incubating is for about 2 hours at a pH value of about pH 5 or about pH 4 at a temperature of about 4° C.

18. The method according to claim 1, characterized in that the incubating is for about 2 hours to about 48 hours at a pH value of about pH 5 or of about pH 4 at a temperature of about 4° C.

Description

DESCRIPTION OF THE FIGURES

[0074] FIG. 1 Host cell DNA content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

[0075] FIG. 2 Host cell protein content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

[0076] FIG. 3 Antibody content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein.

[0077] FIG. 4 Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

[0078] FIG. 5 Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

[0079] FIG. 6 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein.

[0080] FIG. 7 Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

[0081] FIG. 8 Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

[0082] FIG. 9 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein.

[0083] FIG. 10 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

[0084] FIG. 11 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

[0085] FIG. 12 Antibody content in an anti-HERS antibody fermentation culture supernatant obtained with a method as reported herein depending on the pH value and the incubation time.

EXAMPLE 1

Materials and Methods

Antibody

[0086] The herein reported method is exemplified with an anti-EGFR antibody as reported in WO 2008/017963.

[0087] Another exemplary immunoglobulin is an anti-PLGF antibody as reported in WO 2006/099698.

[0088] Another exemplary immunoglobulin is an anti-P-selectin antibody as reported in WO 2005/100402.

[0089] Another exemplary immunoglobulin is an anti-HERS antibody as reported in PCT/EP2010/070062.

DNA

[0090] The residual DNA concentration was measured by Q-PCR (quantitative polymerase chain reaction). Therefore, the samples were incubated at high temperatures to denature the DNA in the sample. The DNA was captured from solution by a Silica matrix and eluted therefrom with buffer according to manufacturer's instructions. For the extraction a QIAcube robot was used including the QIAamp Viral RNA Kit (both from Qiagen, Hilden, Germany). Therefore the sample, lysis buffer and carrier RNA were combined and incubated for 10 minutes. Ethanol was added to each tube and the column of the QIAamp Viral RNA Kit was loaded with the sample-ethanol solution and centrifuged. Afterwards a wash solution was applied to the columns and again the column was centrifuged.

[0091] Thereafter a different wash solution was applied to the column and the column was centrifuged again. After the elution buffer was applied the column is centrifuged twice.

[0092] For the quantification of the DNA a LightCycler 2.0 is used (Roche Diagnostics GmbH, Mannheim, Germany). In Table 1 the PCR parameters are outlined.

TABLE-US-00001 TABLE 1 PCR parameters. cycles step analysis mode temperature hold time 1 preincubation none 40° C. 10 min. 95° C. 10 min. 45 amplification quantification 95° C. 10 min. 60° C. 30 sec. 69° C. 1 sec. 1 cooling none 37° C. 30 sec.

[0093] During the procedure a DNA strands marked with dye binds to the DNA single strands. During the amplification the fluorescence increases proportional to the quantity of DNA.

HCP

[0094] The walls of the wells of a micro titer plate are coated with a mixture of serum albumin and Streptavidin. A goat derived polyclonal antibody against HCP is bound to the walls of the wells of the micro titer plate. After a washing step different wells of the micro titer plate are incubated with a HCP calibration sequence of different concentrations and sample solution. After the incubation not bound sample material is removed by washing with buffer solution. For the detection the wells are incubated with an antibody peroxidase conjugate to detect bound host cell protein. The fixed peroxidase activity is detected by incubation with ABTS and detection at 405 nm.

SEC

[0095] The chromatography was conducted with a Tosoh Haas TSK 3000 SWXL column on an ASI-100 HPLC system (Dionex, Idstein, Germany) The elution peaks were monitored at 280 nm by a UV diode array detector (Dionex). After dissolution of the concentrated samples to 1 mg/ml the column was washed with a buffer consisting of 200 mM potassium dihydrogen phosphate and 250 mM potassium chloride pH 7.0 until a stable baseline was achieved. The analyzing runs were performed under isocratic conditions using a flow rate of 0.5 ml/min over 30 minutes at room temperature. The chromatograms were integrated manually with Chromeleon (Dionex, Idstein, Germany).

IEC

[0096] With the Ion Exchange Chromatography the charge heterogeneity of the protein was analyzed. A cation exchange Dionex ProPac chromatography column was used on a Dionex Chromeleon HPLC system.

Protein Determination

[0097] A chromatographic method was used to quantify the amount of antibody present in a sample. A Poros A column was used that binds the Fc-region of the antibody. The antibody binds to the column and is subsequently eluted by low pH conditions. Protein concentration was determined by determining the optical density (OD) at 280 nm, with a reference wavelength of 320 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

EXAMPLE 2

Procedure

[0098] The samples were obtained directly from the cultivation medium of the respective antibody secreting cell lines. After removal of cells and cell debris the cultivation supernatant was divided into several aliquots of 200 ml each and adjusted to a pH value of pH 4.0, pH 5.0, and pH 6.0, respectively by adding 10% or 20% (v/v) acetic acid. In the reference aliquot the pH value was adjusted if required to approximately pH 7. The aliquots were stored at 4° C. and samples were taken from each aliquot after 2, 24 and 48 hours.

[0099] During storage different levels of precipitate formation were observed at the different pH values. The precipitate was removed by sedimentation. Alternatively, the precipitate can be separated by filtration or centrifugation.

[0100] After removal of the precipitate the clear liquid phase was analyzed for host cell DNA content, host cell protein (HCP) content and protein amount.

TABLE-US-00002 TABLE 2 Host cell DNA content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 7.36 7.02 3.19 8.30 24 7.63 6.59 2.30 8.88 48 7.71 6.95 7.31 6.74

TABLE-US-00003 TABLE 3 Host cell protein content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 528 432 343 457 24 477 471 318 334 48 489 467 426 343

TABLE-US-00004 TABLE 4 Antibody content in an anti-EGFR antibody fermentation culture supernatant obtained with a method as reported herein. protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2268 2307 2207 1530 24 2332 2305 2205 1521 48 2321 2316 2231 1572

TABLE-US-00005 TABLE 5 Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 9.45 7.81 3.11 7.81 24 9.61 8.16 2.30 8.26 48 9.93 8.07 0.84 7.44

TABLE-US-00006 TABLE 5a Host cell DNA content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 5543 3007 286 846 24 2750 1828 65 58 48 3484 1504 103 49

TABLE-US-00007 TABLE 5b Host cell DNA content in the sediment of an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell DNA [μg/g] time [h] pH 5.0 pH 4.0 2 85395 223104 24 183908 242948 48 145992 229317

TABLE-US-00008 TABLE 6 Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1258 1176 276 390 24 1197 1151 205 363 48 1297 1069 178 357

TABLE-US-00009 TABLE 6a Host cell protein content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 983 1051 787 292 24 784 869 501 209 48 831 874 503 210

TABLE-US-00010 TABLE 6b Host cell protein content in the sediment of an anti- PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell protein [mg/g] time [h] pH 5.0 pH 4.0 2 1693 1299 24 2236 1667 48 2024 1021

TABLE-US-00011 TABLE 7 Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1410 1393 1360 644 24 1410 1395 1358 603 48 1410 1394 1356 567

TABLE-US-00012 TABLE 7a Antibody content in an anti-PLGF antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1003 1051 1012 1023 24 1022 1045 1023 1067 48 1016 1044 1045 1045

TABLE-US-00013 TABLE 8 Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). host cell DNA [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 11.38 5.85 2.55 13.35 24 11.52 5.94 1.31 6.85 48 11.22 5.69 1.66 87.40

TABLE-US-00014 TABLE 8a Host cell DNA content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 22068 10228 1 404 24 18250 7953 74 93 48 19305 6280 46 275

TABLE-US-00015 TABLE 8b Host cell DNA content in the sediment of an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 65945 79413 24 — — 90656 41701 48 393649 139806 10126 159270

TABLE-US-00016 TABLE 9 Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). host cell protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 218 209 212 281 24 244 384 208 215 48 214 254 144 1877

TABLE-US-00017 TABLE 9a Host cell protein content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 181 192 120 46 24 212 199 103 77 48 202 207 73 77

TABLE-US-00018 TABLE 9b Host cell protein content in the sediment of an anti- P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 290 367 24 — — 693 129 48 110 108 279 365

TABLE-US-00019 TABLE 10 Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (1.sup.st data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 1880 1880 1859 1094 24 1884 1885 1867 1399 48 1890 1881 1865 127

TABLE-US-00020 TABLE 10a Antibody content in an anti-P-selectin antibody fermentation culture supernatant obtained with a method as reported herein (2.sup.nd data set). protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2950 2933 2929 2231 24 2948 2867 2900 2157 48 3051 2981 2978 2077

TABLE-US-00021 TABLE 11 Host cell DNA content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 12333 15716 745 65 24 8053 19124 201 14 48 7703 15473 52 13

TABLE-US-00022 TABLE 11a Host cell DNA content in the sediment of an anti- HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell DNA [μg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 61749 57288 24 — — 67944 48404 48 304107 186101 71909 25617

TABLE-US-00023 TABLE 12 Host cell protein content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 522 432 186 40 24 488 384 141 31 48 476 346 115 87

TABLE-US-00024 TABLE 12a Host cell protein content in the sediment of an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. host cell protein [mg/g] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 — — 770 465 24 — — 612 424 48 1167 986 726 275

TABLE-US-00025 TABLE 13 Antibody content in an anti-HER3 antibody fermentation culture supernatant obtained with a method as reported herein. protein [μg/ml] time [h] reference pH 6.0 pH 5.0 pH 4.0 2 2992 3035 3047 2928 24 3005 3038 2997 2989 48 3012 3044 3003 2625