Immunoglobulin purification
11261238 · 2022-03-01
Assignee
Inventors
Cpc classification
International classification
A61K39/395
HUMAN NECESSITIES
C07K16/00
CHEMISTRY; METALLURGY
C07K16/24
CHEMISTRY; METALLURGY
Abstract
The current invention reports a method for purifying an immunoglobulin, wherein the method comprises applying an aqueous, buffered solution comprising an immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby the immunoglobulin in monomeric form does not bind to the cation exchange material, and recovering the immunoglobulin in monomeric form from the solution after the contact with the cation exchange material.
Claims
1. A method for obtaining an immunoglobulin in monomeric form from a solution comprising the immunoglobulin in monomeric and in aggregated form, comprising the steps of: 1) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an affinity column under conditions whereby said immunoglobulin binds to said affinity column, and recovering said immunoglobulin in monomeric and in aggregated form from said affinity column; 2) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an anion exchange material under conditions whereby said immunoglobulin does not bind to said anion exchange material, and recovering said immunoglobulin in monomeric and in aggregated form from said solution after the contact with said anion exchange material; and 3) applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby said immunoglobulin in monomeric form does not bind to said cation exchange material, and recovering said immunoglobulin in monomeric form from said solution after the contact with said cation exchange material, wherein: said aggregated form is an immunoglobulin molecule associated either covalently or non-covalently with a least one additional immunoglobulin molecule, said anion exchange material is a chromatography material comprising only anionic charged groups as chromatographically active substituents, and said cation exchange material is a chromatography material comprising only cationic charged groups as chromatographically active substituents.
2. The method according to claim 1, characterized in that said step 3) is a chromatographic step operated in flow-through mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to a cation exchange material under conditions whereby said immunoglobulin in monomeric form does not bind to said cation exchange material, and recovering said immunoglobulin in monomeric form from said solution after the contact with said cation exchange material.
3. The method according to claim 2, characterized in that said step 2) is a chromatographic step operated in flow through-mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an anion exchange material under conditions whereby said immunoglobulin does not bind to said anion exchange material, and recovering said immunoglobulin in monomeric and in aggregated form from said solution after the contact with said anion exchange material.
4. The method according to claim 3, characterized in that said step 1) is a chromatographic step operated in bind-and-elute mode comprising applying an aqueous, buffered solution comprising said immunoglobulin in monomeric and in aggregated form to an affinity column under conditions whereby said immunoglobulin binds to said affinity column, and recovering said immunoglobulin in monomeric and in aggregated form from said affinity column.
5. The method according to claim 1, characterized in that in said step 3) the cation exchange material is a membrane cation exchange material.
6. The method according to claim 5, characterized in that said membrane cation exchange material is a polyethersulfone based membrane or a regenerated cellulose based membrane modified with sulfonic acid groups or carboxymethyl groups.
7. The method according to claim 1, characterized in that said aqueous, buffered solution of step 3) has a pH value of from pH 5 to pH 8.
8. The method according to claim 1, characterized in that said aqueous, buffered solution of step 3) has a conductivity of from 1.0 to 15.0 mS/cm.
9. The method according to claim 8, characterized in that said aqueous, buffered solution of step 3) has a conductivity of from 4.0 to 10.0 mS/cm.
10. The method according to claim 1, characterized in that said recovering said immunoglobulin in monomeric form from the flow-through is by a method selected from precipitation, salting out, ultrafiltration, diafiltration, lyophilization, affinity chromatography, or solvent volume reduction to obtain a concentrated solution.
11. The method according to claim 10, characterized in that said recovering is by ultrafiltration, lyophilization, or solvent volume reduction.
12. The method according to claim 1, characterized in that of said immunoglobulin obtained from the flow-through of the membrane cation exchange material at least 95% of the immunoglobulin is in monomeric from.
13. The method according to claim 1, characterized in that at least 90% of the immunoglobulin in monomeric form does not bind to the cation exchange material.
14. The method according to claim 1, characterized in that said aqueous, buffered solution is a solution comprising phosphoric acid or salts thereof, citric acid or salts thereof, or histidine or salts thereof.
15. The method according to claim 1, characterized in that said aqueous, buffered solution comprises sodium chloride or potassium chloride.
16. The method according to claim 1, characterized in that the sum of pH value and conductivity in mS/cm of the aqueous, buffered solution in step 3) is in the range of from 9 to 18.
17. The method according to claim 16, characterized in that said sum is in the range of from 10 to 15.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
(6) Materials and Methods:
(7) Conditioned Protein a Eluate:
(8) An anti-(IL-13Rα1) antibody (hereinafter referred to as mAb IL13, see e.g. WO 2006/072564) and an anti-Her2 antibody (hereinafter referred to as mAb Her2, see e.g. U.S. Pat. No. 5,677,171) were purified in a first step with a protein A affinity chromatography.
(9) The mAb IL13 was eluted from the protein A column under acidic conditions (3.5 mM hydrochloric acid, pH value 2.7±0.2). Before the filtration step the pH value of the fraction containing the immunoglobulin was adjusted with a concentrated, e.g. 1 M, buffer solution of pH 9.0 (e.g. tris (hydroxymethyl) aminomethane (TRIS) or phosphate buffer) to pH 5.0. This material is referred to in the following as conditioned protein A eluate of mAb IL13.
(10) The mAb Her2 was purified in a first step with a protein A affinity chromatography. Elution from the protein A column is carried out under acidic conditions (10 mM sodium citrate buffer, pH value of 3.0±0.5). Before the filtration step the pH value of the fraction containing the immunoglobulin is adjusted with a concentrated tris (hydroxymethyl)aminomethane (TRIS) buffer to pH 5.6. This material is referred to in the following as conditioned protein A eluate of mAb Her2.
(11) Analytical Methods:
(12) Size Exclusion Chromatography:
(13) TABLE-US-00001 resin: TSK 3000 (Tosohaas) column: 300 × 7.8 mm flow rate: 0.5 ml/min buffer: 200 mM potassium phosphate containing 250 mM potassium chloride, adjusted to pH 7.0 wavelength: 280 nm DNA- see e.g. Merrick, H., and Hawlitschek, G., Biotech threshold- Forum Europe 9 (1992) 398-403 system: Protein A The wells of a micro titer plate are coated with a ELISA: polyclonal anti-protein A-IgG derived from chicken. After binding non-reacted antibody is removed by washing with sample buffer. For protein A binding a defined sample volume is added to the wells. The protein A present in the sample is bound by the chicken antibody and retained in the wells of the plate. After the incubation the sample solution is removed and the wells are washed. For detection are added subsequently a chicken derived polyclonal anti- protein A-IgG-biotin conjugate and a Streptavidin peroxidase conjugate. After a further washing step substrate solution is added resulting in the formation of a colored reaction product. The intensity of the color is proportional to the protein A content of the sample. After a defined time the reaction is stopped and the absorbance is measured. Host cell The walls of the wells of a micro titer plate are coated protein with a mixture of serum albumin and Streptavidin. A (HCP) ELISA: 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.
Example 1
(14) Purification of mAb IL13 to Monomeric Form—Comparison of Conditions
(15) In the purification of mAb IL13 to monomeric form different conditions were evaluated.
(16) The purification method was operated as a chromatographic purification method in flow-through mode. Different conditions for the flow-through purification of the mAb IL13 were evaluated. As membrane cation exchange material the Mustang™ S-Adsorber System (Mustang™ S Coin, 1.5 cm.sup.2 membrane area, Pall Corporation, USA) has been used.
(17) The conditioned protein A eluate had a mAb IL13 concentration of 7.1 g/l with 90.9% of the immunoglobulin in monomeric form and 9.1% of the immunoglobulin in aggregated form. The conditioned protein A eluate has been virus inactivated and filtered through a 0.2 μm pore filter prior to the experiments. After dilution with the corresponding buffer to a protein concentration of approx. 1 mg/ml (a ratio of approx. 1:7 (v/v)), pH and conductivity adjustment, the solutions were applied to the membrane cation exchange material. If necessary the adjustment of pH was performed with potassium hydrogen phosphate or potassium dihydrogenphosphate and conductivity adjustment was done by the addition of KCl or deionized water (with pH of approx. 5.5 and 7.5, respectively). Such a diluted, adjusted, and conditioned protein A eluate is referred to in the following as sample material.
(18) Diversified parameters were conductivity and pH. Observed parameters were yield and purity of the flow-through immunoglobulin in monomeric form of the sample material. The diversified parameters of the sample material are summarized in Table 1.
(19) TABLE-US-00002 TABLE 1 Sample material parameters Sample pH conductivity No. Value buffer [mS/cm] 1 5.5 40 mM potassium phosphate 4.8 2 6.5 40 mM potassium phosphate 4.8 3 7.5 40 mM potassium phosphate 4.8 4 5.5 50 mM potassium phosphate 5.8 5 6.5 50 mM potassium phosphate 5.8 6 7.5 50 mM potassium phosphate 5.8 7 5.5 60 mM potassium phosphate 6.8 8 6.5 60 mM potassium phosphate 6.8 9 7.5 60 mM potassium phosphate 6.8
(20) The flow-through has been analyzed by size exclusion chromatography in order to determine the amount of immunoglobulin in monomeric form and in aggregated form. The second fraction was chosen for analysis because in the beginning of the process (1.sup.st fraction) no stable purification process had established because due to the dead volume of the chromatographic system no steady flow exists and thus an unknown dilution of the 1.sup.st fraction occurs. An exemplary flow-through diagram is depicted in
(21) TABLE-US-00003 TABLE 2 mAb IL13 immunoglobulin in monomeric form (as area percentage of SEC chromatography) and yield with a Mustang ™ S membrane. pH value 5.5 6.5 7.5 conductivity mAb IL13 mAb IL13 mAb IL13 [mS/cm] monomeric form yield monomeric form yield monomeric form yield 4.8 100% 33.6% 100% 82.5% 75% 94.4% 5.8 100% 65.7% 100% 90.9% 0% 100% 6.8 100% 67.9% 99% 91.1% 0% —
(22) The data presented in Table 2 show that conditions suitable for the purification of mAb IL13, i.e. the separation of an immunoglobulin in monomeric form from the aggregated form, i.e. conditions under which the immunoglobulin in monomeric form does not bind to the membrane cation exchange material, with excellent yield can be adjusted, such as e.g. a conductivity of 5.8 mS/cm and a pH of 6.5.
Example 2
(23) Purification of mAb IL13 to Monomeric Form—Comparison of Membranes
(24) The results obtained in Example 1 are generally applicable and have been applied to the membrane cation exchange material Sartobind™ S (75 cm.sup.2 membrane area, Sartorius AG, Göttingen, Germany). The preferred conditions for Mustang™, i.e. conductivity of 5.8 mS/cm at a pH of 6.5, were also applied to the Sartobind™ material. The sample material containing mAb IL13 had a protein concentration of 1.34 mg/ml with 94.8% of the immunoglobulin in monomeric form and 5.2% of the immunoglobulin in aggregated form. The sample material was applied to the membrane as reported in Example 1. The results of the purification process are summarized in Table 3.
(25) TABLE-US-00004 TABLE 3 mAb IL13 immunoglobulin in monomeric and aggregated form (as area percentage of SEC chromatography) and yield with a Sartobind ™ S membrane. Sample mAb IL13 in amount mAb IL13 in monomeric aggregated form in of form in flow-through flow-through Sample mAb Sample total concentration amount amount No. IL13 volume area percentage yield area percentage 1 14.71 mg 11 ml 0.029 mg/ml 100% 1.02 mg 6.9% bdl — 2 14.71 mg 11 ml 0.51 mg/ml 99% 5.59 mg 38.0% 1.00% 0.06 mg 3 14.71 mg 11 ml 0.89 mg/ml 97.6% 9.54 mg 64.9% 2.45% 0.24 mg 4 14.71 mg 11 ml 0.94 mg/ml 97.5% 10.03 mg 68.2% 2.53% 0.26 mg 5 14.71 mg 11 ml 0.94 mg/ml 97.8% 10.11 mg 68.7% 2.17% 0.22 mg 6 14.71 mg 11 ml 0.94 mg/ml 97.9% 10.16 mg 69.1% 2.07% 0.22 mg 7 14.71 mg 11 ml 0.95 mg/ml 97.5% 10.14 mg 68.9% 2.54% 0.26 mg Average 14.71 mg 11 ml 0.94 mg/ml 97.7% 10.11 mg 68.7% 2.33% 0.24 mg of samples 4 to 7 bdl = below detection limit The same sample material was also applied to a Sartobind ™ C membrane. The results are presented in Table 4 and an exemplary size exclusion chromatogram of the 3.sup.rd fraction is shown in FIG. 3.
(26) TABLE-US-00005 TABLE 4 mAb IL13 immunoglobulin in monomeric and aggregated form (as area percentage of SEC chromatography) and yield with a Sartobind ™ C membrane. Sample mAb IL13 in amount mAb IL13 in monomeric aggregated form in of form in flow-through flow-through Sample mAb Sample total concentration amount amount No. IL13 volume area percentage yield area percentage 1 14.71 mg 11 ml 0 mg/ml bdl — — bdl — 2 14.71 mg 11 ml 0.033 mg/ml 100% 0.36 mg 2.4% bdl — 3 14.71 mg 11 ml 0.56 mg/ml 100% 6.15 mg 41.8% bdl — 4 14.71 mg 11 ml 0.87 mg/ml 99.4% 9.54 mg 64.9% 0.65% 0.06 mg 5 14.71 mg 11 ml 0.93 mg/ml 99.3% 10.20 mg 69.3% 0.66% 0.07 mg 6 14.71 mg 11 ml 0.96 mg/ml 98.9% 10.43 mg 70.9% 1.13% 0.12 mg 7 14.71 mg 11 ml 0.98 mg/ml 98.9% 10.66 mg 72.5% 1.08% 0.12 mg Average 14.71 mg 11 ml 0.94 mg/ml 99.1% 10.21 mg 69.4% 0.88% 0.09 mg of samples 4 to 7 bdl = below detection limit — = not determined By using the purification conditions determined with one membrane material in all experiments, all cationic membrane adsorbers demonstrated will have the capability to remove aggregates and to obtain monomeric IgG in flow-through mode operation with the same conditions.
Example 3
(27) Purification of mAb Her2 to Monomeric Form—Comparison of Membranes
(28) The conditioned protein A eluate had a mAb Her2 concentration of 7.61 g/l with 98.8% purity. The immunoglobulin in aggregated form was produced/obtained by heating of the mAb Her2 solution to 37° C. for 3 days. The solution contained after the heat treatment 99.0% of the immunoglobulin in monomeric form and 1.0% of the immunoglobulin in aggregated form without considering low molecular weight substances present in the heat treated sample. The conditioned protein A eluate had been virus inactivated and filtered through a 0.2 μm pore size filter prior to the experiments. After dilution to protein concentration of 1.03 mg/ml, pH and conductivity adjustment, the solution had been applied to the membrane cation exchange materials Sartobind™ S and C, respectively. The adjustment of conductivity was done by the addition of NaCl (5 mol/l). The results are shown in Tables 5 and 6.
(29) TABLE-US-00006 TABLE 5 mAb Her2 immunoglobulin in monomeric and aggregated form (as area percentage of SEC chromatography) and yield with a Sartobind ™ S membrane. Sample mAb Her2 in amount mAb Her2 in monomeric aggregated form in of form in flow-through flow-through Sample mAb Sample total concentration amount amount No. Her2 volume area percentage yield area percentage 1 11.35 mg 11 ml 0.053 mg/ml 40.4% 0.23 mg 0.02% bdl — 2 11.35 mg 11 ml 0.600 mg/ml 99.2% 6.47 mg 57.0% bdl — 3 11.35 mg 11 ml 0.908 mg/ml 99.8% 9.86 mg 86.9% 0.02% 0.002 mg 4 11.35 mg 11 ml 0.977 mg/ml 99.9% 10.60 mg 93.4% 0.03% 0.003 mg 5 11.35 mg 11 ml 0.999 mg/ml 99.8% 10.83 mg 95.4% bdl — 6 11.35 mg 11 ml 1.013 mg/ml 99.9% 10.99 mg 96.8% bdl — 7 11.35 mg 11 ml 1.019 mg/ml 99.8% 10.04 mg 88.5% 0.05% 0.006 mg 8 11.35 mg 11 ml 1.023 mg/ml 99.9% 11.10 mg 97.8% 0.08% 0.009 mg 9 11.35 mg 11 ml 1.025 mg/ml 99.8% 11.12 mg 98.0% 0.14% 0.016 mg 10 11.35 mg 11 ml 1.024 mg/ml 99.8% 11.11 mg 97.9% 0.17% 0.019 mg 11 11.35 mg 11 ml 1.025 mg/ml 99.7% 11.11 mg 97.9% 0.18% 0.020 mg 12 11.35 mg 11 ml 1.027 mg/ml 99.7% 11.13 mg 98.1% 0.23% 0.026 mg 13 11.35 mg 11 ml 1.028 mg/ml 99.7% 11.13 mg 98.1% 0.24% 0.027 mg 14 11.35 mg 11 ml 1.028 mg/ml 99.6% 11.13 mg 98.1% 0.30% 0.034 mg 15 11.35 mg 11 ml 1.027 mg/ml 99.6% 11.30 mg 99.6% 0.32% 0.036 mg Average 11.35 mg 11 ml 1.018 mg/ml 99.8% 10.97 mg 96.7% 0.14% 0.016 mg of samples 4 to 15 bdl = below detection limit — = not determined
(30) TABLE-US-00007 TABLE 6 mAb Her2 immunoglobulin in monomeric and aggregated form (as area percentage of SEC chromatography) and yield with a Sartobind ™ C membrane. Sample mAb Her2 in amount mAb Her2 in monomeric aggregated form in of form in flow-through flow-through Sample mAb Sample total concentration amount amount No. Her2 volume area percentage yield area percentage 1 11.35 mg 11 ml 0.082 mg/ml 74.8% 0.67 mg 5.9% bdl — 2 11.35 mg 11 ml 0.656 mg/ml 99.1% 7.07 mg 62.3% bdl — 3 11.35 mg 11 ml 0.861 mg/ml 99.7% 9.32 mg 82.1% bdl — 4 11.35 mg 11 ml 0.929 mg/ml 99.7% 10.06 mg 88.6% 0.04% 0.004 mg 5 11.35 mg 11 ml 0.965 mg/ml 99.7% 10.45 mg 92.1% 0.09% 0.01 mg 6 11.35 mg 11 ml 0.992 mg/ml 99.7% 10.47 mg 92.2% 0.15% 0.015 mg 7 11.35 mg 11 ml 1.004 mg/ml 99.8% 10.88 mg 95.9% 0.14% 0.015 mg 8 11.35 mg 11 ml 1.014 mg/ml 99.7% 10.98 mg 96.7% 0.24% 0.027 mg 9 11.35 mg 11 ml 1.015 mg/ml 99.7% 10.99 mg 96.8% 0.29% 0.032 mg 10 11.35 mg 11 ml 1.017 mg/ml 99.6% 11.00 mg 96.9% 0.36% 0.04 mg 11 11.35 mg 11 ml 1.019 mg/ml 99.6% 11.03 mg 97.2% 0.43% 0.047 mg 12 11.35 mg 11 ml 1.021 mg/ml 99.5% 11.04 mg 97.3% 0.41% 0.046 mg 13 11.35 mg 11 ml 1.022 mg/ml 99.5% 11.05 mg 97.4% 0.42% 0.047 mg 14 11.35 mg 11 ml 1.024 mg/ml 99.5% 11.07 mg 97.5% 0.51% 0.055 mg 15 11.35 mg 11 ml 1.024 mg/ml 99.5% 11.07 mg 97.5% 0.47% 0.051 mg Average 11.35 mg 11 ml 1.0 mg/ml 99.6% 10.84 mg 95.5% 0.30% 0.032 mg of samples 4 to 15 bdl = below detection limit — = not determined An exemplary size exclusion chromatography (SEC) analysis of fraction 4 of the flow-through of a Sartobind C membrane is shown in FIG. 4.
(31) It can be summarized from the above that the sum of pH value and conductivity in mS/cm is preferably in the range from 9 to 18, more preferably in the range from 10 to 15.
Example 4
(32) Analysis of Protein a, DNA and HCP Content.
(33) The fractions obtained in Example 2 have been analyzed for protein A-, DNA-, and HCP-content. The results are given in Tables 7 and 8.
(34) The protein A-content in the solution prior to the application to the Sartobind™ S membrane was 2.6 ng/ml, the DNA-content was <0.3 pg/mg, and the host cell protein (HCP)-content was 1791 ng/mg.
(35) TABLE-US-00008 TABLE 7 Protein A-, DNA-, and HCP-content of fractions containing mAb IL13 immunoglobulin obtained with a Sartobind ™ S membrane. Sample Protein Sample volume concentration protein A DNA HCP No. [ml] [mg/ml] [ng/mg] [pg/mg] [ng/mg] 1 11 0.029 <4.1 <138 28 2 11 0.513 0.5 <0.8 <118 3 11 0.889 <0.3 <0.4 <202 4 11 0.935 0.7 <0.4 255 5 11 0.939 — <0.4 — 6 11 0.943 — <0.4 — — = not determined The protein A-content in the solution prior to the application to the Sartobind ™ C membrane was 3.0 ng/ml, the DNA-content was <0.4 pg/mg, and the host cell protein (HCP)-content was 5250 ng/mg.
(36) TABLE-US-00009 TABLE 8 Protein A-, DNA-, and HCP-content of fractions containing mAb IL13 immunoglobulin obtained with a Sartobind ™ C membrane. Sample Protein Sample volume concentration protein A DNA HCP No. [ml] [mg/ml] [ng/mg] [pg/mg] [ng/mg] 1 11 0.033 <4.2 <13.3 155 2 11 0.559 <0.2 <0.7 1036 3 11 0.873 0.3 <0.5 989 4 11 0.933 0.7 <0.4 1366 5 11 0.959 n.b. n.b. n.b. 6 11 0.979 n.b. n.b. n.b.