Method for the purification of antibodies

Abstract

A method for the purification of immunoglobulins by ion exchange chromatography is described. The chromatographic method uses a weak ion exchange resin and a single step elution process for the purification of an immunoglobulin. Additionally a method for the determination of the salt concentration for the single step elution of an immunoglobulin from an ion exchange resin is described.

Claims

1. A method for purifying a monomeric monoclonal anti-HER2 antibody from aggregates thereof, wherein the method comprises: a) purifying a monoclonal anti-HER2 antibody by protein A affinity chromatography; b) providing a solution comprising the monoclonal anti-HER2 antibody of step a); c) bringing the solution of step b) and a weak cation exchange material in contact under conditions whereby the monoclonal anti-HER2 antibody binds to the weak cation exchange material; and d) recovering the monomeric monoclonal anti-HER2 antibody from the weak cation exchange material in a single step by using a second solution comprising a buffer substance and a salt wherein the conductivity of the second solution is increased by changing one condition all at once from a starting value to a final value so as to obtain the monomeric monoclonal anti-HER2 antibody purified from aggregates thereof, wherein the buffer substance and salt are the same and the monoclonal anti-HER2 antibody has an isoelectric point (p1) of 6.0 or higher.

2. The method according to claim 1, wherein the buffer substance of step d) is selected from the group consisting of citric acid, a salt of citric acid, phosphoric acid, and a salt of phosphoric acid.

3. The method according to claim 1, wherein the method is a chromatographic or a batch method.

4. The method according to claim 1, wherein the salt of step d) is selected from the group consisting of sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, salts of citric acid, salts of phosphoric acid, and mixtures thereof.

5. The method according to claim 2, wherein the method is a chromatographic or a batch method.

6. The method according to claim 1, wherein the pH is kept constant in the single step.

7. The method according to claim 2, wherein the pH is kept constant in the single step.

8. The method according to claim 3, wherein the pH is kept constant in the single step.

9. The method according to claim 1, wherein the salt in elution step d) is added at the same time as the buffer substance.

10. The method according to claim 1, wherein the weak cation exchange material is a carboxy-methyl weak cation exchange material.

11. The method according to claim 1, wherein the monoclonal anti-HER2 antibody is a member of the immunoglobulin class G.

12. The method according to claim 1, wherein the second solution in the recovery step d) has a pH value of from pH 3.0 to pH 7.0.

13. The method according to claim 2, wherein the weak cation exchange material is a carboxy-methyl weak cation exchange material.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Single step elution of anti IL-1R antibody from strong cation exchange resin SP-Sepharose; monomeric and aggregated forms of the receptor antibody are not separated and elute as one peak.

(2) FIG. 2 Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A.

(3) FIG. 3 Linear gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl with sodium citrate at pH 5.0; monomeric and aggregated forms of the receptor antibody are partially separated and elute at a sodium citrate starting concentration of 15 mM as a main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising a mixture of monomeric form, aggregated forms of the immunoglobulin, and protein A. The SEC analysis of the two fractions is inserted into the ion exchange chromatogram. In the main peak aggregates are absent. In contrast in the second peak aggregated forms of the immunoglobulin are present.

(4) FIG. 4 Three step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl with sodium citrate at pH 5.0; monomeric and aggregated forms of the receptor antibody are partially separated and elute as a main peak comprising the monomeric form of the immunoglobulin at a sodium citrate concentration of 34 mM and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A at a sodium citrate concentration of 44 mM.

(5) FIG. 5 Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 5.5; monomeric and aggregated forms of the receptor antibody are separated and elute as a main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A.

(6) FIG. 6a Elution profile on a CM-Sepharose fast flow; two peaks can be identified: a main peak, corresponding to the monomeric anti IL-1R antibody, and a smaller second peak, that contains mainly aggregates and other impurities.

(7) FIG. 6b Elution profile of anti IL-1R antibody on a SP-Sepharose fast flow; one peak can be identified which contains monomeric immunoglobulin, aggregates and other impurities which have not been separated on this column.

(8) FIG. 7a Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 100 mM sodium chloride at pH 5.5.

(9) FIG. 7b Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 5.5.

(10) FIG. 7c Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 200 mM sodium chloride at pH 5.5.

(11) FIG. 8a Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 4.0.

(12) FIG. 8b Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 6.0.

(13) FIG. 9a Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; size exclusion chromatography

(14) (SEC) of the starting material showing both monomeric and aggregated forms of the immunoglobulin.

(15) FIG. 9b Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as other protein. In this figure the size exclusion chromatography of the first (main) peak is shown. Only one peak is eluted from the SEC-column, which is the monomeric immunoglobulin.

(16) FIG. 9c Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as other protein. In this figure the size exclusion chromatography of the second peak is shown. In the chromatogram at least three peaks can be seen, equivalent to monomeric and aggregated forms of the immunoglobulin and other protein.

(17) FIG. 10 Single step elution of anti-HER-2 antibody from strong cation exchange resin SP-Sepharose; monomeric and aggregated forms of the antibody are not separated and elute as one peak.

(18) FIG. 11 Linear gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with sodium chloride in sodium citrate buffer at pH 5.5; monomeric and aggregated forms of the antibody are partially separated and elute with a starting concentration of 80 mM sodium chloride as one main peak comprising the monomeric form of the immunoglobulin; the monomeric and aggregated forms elute as mixture in the tailing of the main peak together with protein A.

(19) FIG. 12 Three step gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with sodium chloride in sodium citrate buffer at pH 5.5; monomeric and aggregated forms of the antibody are separated and elute as one main peak comprising the monomeric form of the immunoglobulin at a sodium chloride concentration of 80 mM and as a second peak comprising monomeric and aggregated forms of the immunoglobulin as well as protein A at a sodium chloride concentration of 100 mM.

(20) FIG. 13 Single step gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with 80 mM sodium chloride at pH 5.5; the monomeric form is eluted free of aggregated forms; the aggregated forms elute after a second sodium chloride step to 120 mM as a second defined peak.

(21) FIG. 14 Single step gradient elution of anti-HER-2 antibody from strong cation exchange resin SP-Sepharose with 80 mM sodium chloride at pH 5.5; two peaks are obtained: peak 1 contains only monomeric Herceptin®, peak 2, which is eluted after a second sodium chloride step to 120 mM, contains a high amount of monomeric Herceptin® and aggregates; the yield of Herceptin® in its monomeric form is less than 60%.

EXPERIMENTAL PART

(22) Material:

(23) An IgG4 immunoglobulin anti IL-1R antibody (hereinafter referred to as immunoglobulin, WO 2005/023872) was purified in a first step with a protein A affinity chromatography. Elution from the protein A column was carried out under acidic conditions (10 mM sodium citrate buffer, pH value 3.0±0.5). 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. The protein A eluate is a solution with a protein concentration between 5 mg/ml and 15 mg/ml and is buffered with sodium citrate. This material is referred to in the following as conditioned protein A eluate, which is prepared for loading onto a cationic exchange resin.

Example 1

(24) In this comparative example an ion exchange chromatography with a strong cation exchange resin and single step elution is described.

(25) Chromatographic Conditions: Resin: SP-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0 Loading: max. 20 g protein/L gel matrix Wash step: 10 mM sodium citrate buffer, adjusted to pH 5.0 Elution: 25 mM sodium citrate buffer with 100 mM sodium chloride, adjusted to pH 5.0

(26) The conditioned protein A eluate was applied to a chromatography column containing a strong cation exchange resin (SP-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value was kept constant and the conductivity was varied (increased) by the addition of sodium chloride.

(27) In FIG. 1 the elution chromatogram of the cation exchange chromatography of the anti IL-1R antibody on the strong cation exchange resin SP-Sepharose is presented. The elution is a single step replacement elution with sodium chloride without altering the pH value of the chromatographic system. The monomeric and aggregated immunoglobulin molecules are not separated and thus with this method no purification by reduction of the aggregate content in the eluate compared to the loaded material can be obtained.

Example 2

(28) In this example an ion exchange chromatography with a weak cation exchange resin and single step elution is described.

(29) To achieve a separation of monomeric and aggregated forms of an immunoglobulin, a weak cation exchange resin was employed. By using this kind of resin an increase of the conductivity by a step elution is accompanied by a particular pH shift on the resin (even when the pH of the eluting buffer remains constant). This effect facilitates the discrimination e.g. between monomeric immunoglobulins and aggregated forms. Furthermore, other impurities, like traces of host cell proteins or protein A, can efficiently be separated from the monomeric mean fraction, without significant loss in yield.

(30) Chromatographic Conditions: Resin: CM-Toyopearl Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0 Elution: 25 mM sodium citrate buffer with 100 mM sodium chloride, adjusted to pH 5.0

(31) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value in the mobile phase was kept constant and the conductivity was varied by the addition of sodium chloride.

(32) In FIG. 2 the elution profile with the same chromatographic conditions as in example 1 but this time with a weak cation exchange resin, CM-Toyopearl, is presented. Herein a second peak as shoulder of the main peak appears. This separation behavior is different to that with a strong cation exchange resin, such as SP-Sepharose. An analysis of fractions corresponding to the main peak and to the second shoulder peak showed a significant amount of aggregates to be present in the shoulder peak fraction. No aggregates were detectable in the main peak fractions (see also FIGS. 9a to c).

Example 3

(33) Optimization of the Chromatographic Method—First Step: Linear Concentration Gradient.

(34) To optimize the cation exchange chromatography on a weak cation exchange material an optimization procedure, which is consisting of three steps, was put into practice:

(35) The first step is a chromatography using a linear concentration gradient of the buffer salt, sodium citrate. Just as well is it possible to keep the concentration of the buffer salt constant and to admix a linearly increasing concentration of a salt causing the elution of the immunoglobulin. In both cases is the conductivity of the solution increased without an alteration of the pH value of the mobile phase. Salts suitable for the elution are e.g. sodium chloride, sodium sulphate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate, citric acid and salts thereof as well as mixtures of these components. The concentrations of from 10 mM to 500 mM, which are applied, are adjusted accordingly to set conductivity in the range of from about 1 milli S/cm to about 50 milli S/cm.

(36) Chromatographic Conditions: Resin CM-Toyopearl Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0 Elution: linear gradient; from 10 mM sodium citrate buffer, adjusted to pH 5.0, to 100 mM sodium citrate buffer, adjusted to pH 5.0

(37) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a linear gradient elution method, whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was raised linearly from 10 mM to 100 mM over 40 column volumes. After the final sodium citrate concentration was reached the elution was continued for an additional 40 column volumes.

(38) In FIG. 3 the chromatogram of the linear buffer gradient elution of the anti IL-1R antibody is presented. The monomeric and the aggregated form of the immunoglobulin elute in a semi-detached peak starting at a concentration of 15 mM sodium citrate and ending at a concentration of 55 mM sodium citrate.

(39) The recovery of the bound immunoglobulin from the cation exchange resin is depending on the conductivity of the applied solution. Therefore the cations of the buffer salt present in the eluting solution during the recovery step have to be considered as effecting the elution of the immunoglobulin from the cation exchange resin. The conductivity as well as the ionic strength of the mobile phase are effected by the total number of ions in the solution. Thus the number of monovalent cations different from hydrogen in one molecular formula of the employed buffer salt and the employed salt causing the elution have to be considered hereby.

Example 4

(40) Optimization of the Chromatographic Method—Second Step: Three Step Concentration Gradient Elution.

(41) The concentration, at which the elution of the immunoglobulin from the ion exchange resin starts, as determined in example 3, provides the basis for the second optimization step, a three step elution method. The approximate buffer/salt concentrations for the step elution are calculated as follows: the salt concentration of the first elution step is equal to the sum of as first summand the product of the concentration of the salt, at which the elution from the ion exchange column starts as determined with the linear increasing salt gradient, and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt causing the elution and as second summand the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt; the salt concentration of the second elution step is equal to the product of the salt concentration of the first elution step and a factor of between 1.25 and 1.35; the salt concentration of the third elution step is equal to the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70.

(42) The factor included in the calculation of the concentration steps accounts for the interval between the concentration levels and is adjusted depending on the starting concentration. At small starting concentrations, i.e. between 10 mM and 40 mM, the factors are 1.35 and 1.70 respectively, at medium starting concentrations between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at high starting concentrations of more than 70 mM the factors are 1.25 and 1.50 respectively.

(43) The factors define a range that has been determined experimentally. These values are no absolute values but merely a target value. A deviation of 10% is maintainable.

(44) The buffer salt has to be accounted for in the calculation because it is possible as outlined in example 3 that the elution of a protein from an ion exchange resin can be effected by a change of the buffer salt concentration during the chromatography. If the buffer salt concentration is kept constant during the chromatography or is small compared to the stating concentration (≤15% of the salt concentration) it may be neglected during the calculation to reduce complexity.

(45) With a starting concentration of 15 mM sodium citrate, as determined in example 3, consisting of a 10 mM buffer concentration and a 5 mM contribution from the linear gradient, the three steps can be calculated as follows: the target concentration for step 1 is calculated to be 30 mM (5 mM*2+10 mM*2) sodium citrate in detail: 5 mM (starting concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula) plus 10 mM (buffer salt concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula) the target concentration for step 2 is calculated to be 40.5 mM (30 mM*1.35) sodium citrate in detail: 30 mM sodium citrate is the concentration of step 1 multiplied by 1.35 (the starting concentration is 15 mM, therefore as factor 1.35 is selected) the target concentration for step 3 is calculated to be 51 mM (30 mM*1.70) sodium citrate in detail: 30 mM sodium citrate is the concentration of step 1 multiplied by 1.70 (the starting concentration is 15 mM, therefore as factor 1.70 is selected)

(46) Chromatographic Conditions: Resin: CM-Toyopearl Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0 Elution: step 1: 34 mM sodium citrate buffer, adjusted to pH 5.0 step 2: 44 mM sodium citrate buffer, adjusted to pH 5.0 step 3: 54 mM sodium citrate buffer, adjusted to pH 5.0

(47) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a step gradient elution method (=a method wherein the concentration of the elution salt is changed stepwise from a starting value/level to a final value/level), whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was raised from 10 mM as starting condition to 34 mM in the first step, to 44 mM in the second step, and to 54 mM in the final step. After each increase of the salt concentration ten column volumes of the elution buffer were passed through the column prior to the next step. After the final sodium citrate concentration was reached the elution was continued for an additional 10 column volumes.

(48) In FIG. 4 the elution profile of the three step gradient elution of anti IL-1R antibody is presented. The monomeric immunoglobulin elutes in the first step fraction and the aggregates elute in the second step fraction.

Example 5

(49) Optimization of the Chromatographic Method—Third Step: Single Step Elution with Sodium Chloride.

(50) The final step of the optimization procedure is the adaptation to a single step elution method (=a method wherein the concentration of the elution salt is changed at once from a starting value to a final value). For this purpose the pH of the chromatography is raised from 5.0 to 5.5. This pH shift improves the separation from protein A, due that protein A has an isoelectric point below 5.5. Additionally the elution salt is changed from sodium citrate, which is further on used as buffer salt, to sodium chloride. Additional analyses have been carried out (DNA, host cell protein, protein A content, and glycosylation pattern with LC-MS) with the fractions after this chromatographic run.

(51) Chromatographic Conditions: Resin: CM-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate, adjusted to pH 5.5 Elution: 10 mM sodium citrate with 150 mM sodium chloride, adjusted to pH 5.5

(52) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method (=a method wherein the concentration of the elution salt is changed at once from a starting value to a final value), whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and 150 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin.

(53) The elution chromatogram of the single step elution with sodium chloride is presented in FIG. 5. The single step gradient chromatography effects resolution of the main monomeric fraction and the aggregate/protein A fraction. The yield of monomeric immunoglobulin is more than 80%. Even more than 95% yield is possible.

Example 6

(54) Comparison Between the Separation with a Strong Cation Exchange Resin (SP-Sepharose Fast Flow) and a Weak Cation Exchange Resin (CM-Sepharose Fast Flow).

(55) A comparison between the strong SP-Sepharose ff exchanger and CM-Sepharose ff was done. Experiments were performed according to example 5 in duplicates (only one from each column is shown in FIGS. 6a and b) and additional analyses have been carried out (DNA, host cell protein, protein A content, and glycosylation pattern with LC-MS).

(56) Analytical Methods:

(57) TABLE-US-00001 Size Exclusion resin: TSK 3000 (Tosohaas) Chromatography: 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 DNA-threshold- see e.g. Merrick, H., and Hawlitschek, G., Biotech system: Forum Europe 9 (1992) 398-403 Protein The wells of a micro titer plate are coated with a poly- A ELISA: clonal 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 anti- body 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 subse- quently a chicken derived polyclonal anti-protein A- IgG-biotin conjugate and a streptavidin peroxidase conjugate. After a further washing step substrate solu- tion is added resulting in the formation of a colored reaction product. The intensity of the color is propor- tional to the protein A content of the sample. After a defined time the reaction is stopped and the absor- bance is measured. Host cell protein The walls of the wells of a micro titer plate are coated (HCP) ELISA: 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 con- jugate to detect bound host cell protein. The fixed peroxidase activity is detected by incubation with ABTS and detection at 405 nm.

(58) Chromatographic Conditions: Resin: CM-Sepharose; SP-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5 Elution: 10 mM sodium citrate buffer with 150 mM sodium chloride, adjusted to pH 5.5

(59) In FIGS. 6a and 6b a comparison between the elution chromatogram of a weak and a strong cation exchange resin is presented. Using a weak cation exchange resin (FIG. 6a) a separation of the monomeric anti IL-1R antibody from other impurities is achieved. With the strong cation exchange resin (FIG. 6b) no separation is possible under the same conditions. The fractions corresponding to the peaks have been collected and analyzed. The analysis results, which are listed in table 1, show that with the weak cation exchange resin aggregates and other impurities can effectively be depleted from the immunoglobulin preparation.

(60) The data presented in table 1 show that it is possible to separate with a weak cation exchange resin monomeric anti IL-1R antibody from aggregated forms of the antibody. Furthermore DNA- and protein A-impurities can be depleted.

(61) TABLE-US-00002 TABLE 1 Analysis of the eluates: comparison between SP-Sepharose and CM- Sepharose, results of two different separations are presented. conditioned protein A SP-Sepharose eluate CM-Sepharose eluate analyte eluate single peak Peak 1 Peak 2 amount of between 20 31 ng/mg 26 ng/mg  7.5 ng/mg  11 ng/mg 1638 ng/mg  550 ng/mg protein A and 50 ng/mg HCP between 20 ng/mg 3.88 ng/mg   3.98 ng/mg   3.13 ng/mg  3.27 ng/mg   946 ng/mg 1424 ng/mg and 120 ng/mg DNA between 36 pg/mg 16 pg/mg 157 pg/mg 131 pg/mg 1918 pg/mg 1222 pg/mg 2800 and 3500 pg/mg aggregates present present present not not present present present present in high in high amount amount mass no differences were found between SP- and CM-Sepharose analysis

Example 7

Comparative Example—Elution at Different Conductivities

(62) Chromatographic Conditions: Resin: CM-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5 Elution: 10 mM sodium citrate buffer with 100 mM, 150 mM or 200 mM sodium chloride, adjusted to pH 5.5

(63) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and in three different runs 100 mM, 150 mM, and 200 mM sodium chloride respectively were admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin. The elution chromatograms are displayed in FIGS. 7a to c.

(64) Good separations have been obtained using 150 mM sodium chloride and 200 mM sodium chloride as elution salt concentration.

Example 8

Comparative Example—Elution at Different pH Values

(65) Chromatographic Conditions: Resin: CM-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5 Elution: 10 mM sodium citrate buffer with 150 mM sodium chloride, adjusted to pH 4.0, or 6.0

(66) The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase was kept constant at pH 4.0 or 6.0 respectively. The concentration of the buffer salt, sodium citrate, was kept constant, and 150 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin. The elution chromatograms are displayed in FIGS. 8a and b.

(67) At pH 4.0 is the tendency to form aggregates of this immunoglobulin increased. But the CM-Sepharose is able to separate this higher amount of aggregates in two peaks.

Example 9

(68) Chromatographic Separation of a Monoclonal Anti-HER-2 Antibody (WO 99/57134) with a Strong Cation Exchange Resin (SP-Sepharose).

(69) The current invention is further exemplified in the following with Herceptin®, a monoclonal anti-HER-2 antibody.

(70) The purification of Herceptin with a cation exchange chromatography on SP-Sepharose, a strong cation exchange resin, was carried out. Under standard conditions of the current invention, i.e. step elution with e.g. sodium chloride, a separation of monomeric and aggregated forms of the antibody is not effected (FIG. 10).

(71) Chromatographic Conditions: Resin: SP-Sepharose Flow rate: 160 cm/h Equilibration: 25 mM 2-morpholinoethanesulfonic acid, 50 mM sodium chloride, adjusted to pH 5.6 Loading: max. 20 g protein/L gel matrix Wash: 25 mM 2-morpholinoethanesulfonic acid, 50 mM sodium chloride, adjusted to pH 5.6 Elution: 25 mM 2-morpholinoethanesulfonic acid, 95 mM sodium chloride, adjusted to pH 5.6

(72) The monoclonal anti-HER-2 antibody (hereinafter referred to as Herceptin®) 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 antibody is adjusted with a concentrated tris-hydroxymethyl-aminomethane (TRIS) buffer to pH 5.6. The protein A eluate is a solution with a protein concentration between 5 mg/ml and 15 mg/ml and is buffered with sodium citrate.

(73) The conditioned protein A eluate was applied to a chromatography column containing a strong cation exchange resin (SP-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value was kept constant and the conductivity was varied by the (stepwise) increase of the sodium chloride concentration. The elution chromatogram is displayed in FIG. 10.

(74) No separation of monomeric and aggregated forms of the antibody was achieved.

Example 10

(75) Optimization of the Chromatographic Method—First Step: Linear Concentration Gradient.

(76) To improve the separation of the two fractions the separation conditions have been optimized in accordance with the procedure as outlined with the anti IL-1R antibody.

(77) In contrast to the anti IL-1R antibody optimization process a linear gradient of a (elution) salt, i.e. of sodium chloride, was used instead of a gradient of the buffer substance. The chromatogram of the linear sodium chloride gradient elution, which corresponds to the first step of the optimization procedure, is presented in FIG. 11. Analysis confirmed that the tail of the main peak is enriched with aggregated forms of the antibody.

(78) Chromatographic Conditions: Resin CM-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5 Elution: linear gradient; from 10 mM sodium citrate buffer, adjusted to pH 5.5, to 10 mM sodium citrate buffer containing 400 mM sodium chloride, adjusted to pH 5.5

(79) The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a linear gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt was kept constant. The concentration of the elution salt, sodium chloride, was raised linearly from 0 mM to 400 mM over 40 column volumes. The elution chromatogram is displayed in FIG. 11.

(80) The replacement of the strong cation exchange resin by a weak cation exchange resin caused the detachment of a second peak as shoulder of the first main peak. This observation is similar to the observation in case of the anti IL-1R antibody.

(81) The immunoglobulins start to elute from the column at a sodium chloride concentration of 80 mM.

Example 11

(82) Optimization of the Chromatographic Method—Second Step: Three Step Concentration Gradient Elution.

(83) The starting concentration, at which the immunoglobulin starts to elute, as determined in example 10 and as derived from the chromatogram presented in FIG. 11, is 80 mM sodium chloride. For the calculation of the three concentration steps for the second optimization step the buffer concentration can be neglected as it is low and kept constant during the chromatography.

(84) The starting concentration of the sodium chloride is 80 mM and sodium chloride has one cation different from hydrogen in its molecular formula. Accordingly the concentrations for the three step elution are calculated to be 80 mM, 100 mM (=80 mM multiplied with 1.25), and 120 mM (=80 mM multiplied with 1.50) sodium chloride respectively.

(85) Chromatographic Conditions: Resin: CM-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5 Elution: step 1: 10 mM sodium citrate buffer with 80 mM sodium chloride, adjusted to pH 5.5 step 2: 10 mM sodium citrate buffer with 100 mM sodium chloride, adjusted to pH 5.5 step 3: 10 mM sodium citrate buffer with 120 mM sodium chloride, adjusted to pH 5.5

(86) The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a step gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt, sodium citrate, was kept constant. The concentration of the elution salt, sodium chloride, was raised from 0 mM as starting condition to 80 mM in the first step, to 100 mM in the second step, and to 120 mM in the final step. After each increase of the salt concentration ten column volumes of the elution buffer with the specified sodium chloride concentrations were passed through the column prior to the next concentration step. After the final sodium citrate concentration was reached the elution was continued for an additional 10 column volumes. The elution chromatogram is displayed in FIG. 12.

(87) In the three step elution method the monomeric antibody is eluted at the step with a sodium chloride concentration of 80 mM. Size exclusion analysis confirmed that only monomeric antibody is eluted. After the sodium chloride concentration was increased to 100 mM in the second step, the aggregated forms eluted (FIG. 12).

Example 12

(88) Optimization of the Chromatographic Method—Third Step: Single Step Elution with Sodium Chloride.

(89) Chromatographic Conditions: Resin: CM-Sepharose; SP-Sepharose Flow rate: 160 cm/h Equilibration: 10 mM sodium citrate, adjusted to pH 5.5 Loading: max. 20 g protein/L gel matrix Wash: 10 mM sodium citrate, adjusted to pH 5.5 Elution: 10 mM sodium citrate with 80 mM sodium chloride, adjusted to pH 5.5

(90) The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and 80 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer with sodium chloride were passed through the column to elute the bound anti-HER-2 antibody in monomeric form. To affirm the separation of monomeric and aggregated forms of the antibody a second step, which is not necessary for the preparation of monomeric antibodies, to a sodium chloride concentration of 120 mM was performed. After this second increase the aggregated forms of the antibody eluted from the column. The elution chromatogram is displayed in FIG. 13.

(91) If the same method is performed with a strong cation-exchange resin a significant loss of monomeric antibody is observed (yield of approximately 60% only compared to 95% and more on a weak cation exchange column), although the separation of monomeric and aggregated form of the antibody can be seen (FIG. 14).

(92) Applying the conditions suitable for the separation on a weak cation exchange material to a strong cation exchange resin, SP-Sepharose, is not beneficial. Albeit the two fractions can be separated the yield of the monomeric antibody is reduced to 60% or even less.

(93) TABLE-US-00003 TABLE 2 Analysis of the eluates: comparison between SP-Sepharose and CM- Sepharose, results of two different separations are presented. conditioned protein A SP-Sepharose CM-Sepharose analyte eluate Peak 1 Peak 2 Peak 1 Peak 2 amount of  17 ng/mg <3.9 ng/mg.sup.    5.7 ng/mg <3.9 ng/mg  .sup. <3.9 ng/mg   32.2 ng/mg   39.7 ng/mg protein A HCP 3243 ng/mg 49.0 ng/mg 183.4 ng/mg  107.5 ng/mg   126.2 ng/mg  1247 ng/mg  988 ng/mg DNA 1615 pg/mg  830 pg/mg 2635 pg/mg 319 pg/mg   682 pg/mg 10554 pg/mg <9300 pg/mg aggregates 0.97% 0% 32% 0% 0% 15.2% 23.0% (SEC)