WO2002013859A1 - Method of inhibiting antibody-containing solution from coagulating or becoming turbid - Google Patents

Abstract

A method of inhibiting an antibody-containing solution during ultrafiltration from coagulating or becoming turbid, characterized by adding to the antibody-containing solution a surfactant such as polysorbate; and an antibody-containing composition stable to physical stress, characterized by containing 0.00025 to 0.5 wt.% surfactant, e.g., polysorbate, and containing substantially no salts.

Description

 Description Method for suppressing the formation of aggregates or clouding of antibody-containing solution Field of the Invention

 The present invention relates to a method for stabilizing an antibody-containing solution against pressure stress. More specifically, the present invention relates to a method for stabilizing an antibody-containing solution during the ultrafiltration of an antibody-containing solution. How to control. The present invention further relates to an antibody-containing composition stabilized against pressure stress by the above-mentioned means. The present invention is particularly useful in the manufacture of a medicament. Background art

 Antibodies are important and widely used as components of preventive or therapeutic agents for diseases or as components of diagnostic agents. In the production and formulation of antibody drug substances, it may be necessary to ultrafiltrate the antibody-containing solution for various purposes, such as concentrating the antibody-containing solution. During these ultrafiltrations, aggregates or cloudiness may be formed in the antibody-containing solution. It is expected to be caused by insolubilization and aggregation of other contaminants. The use of antibody proteins as therapeutic or diagnostic agents does not produce aggregates or cloudiness, particularly as injections.

Therefore, in the ultrafiltration treatment of the antibody-containing solution, it is important to suppress the formation and clouding of aggregates. Conventionally, however, methods for effectively preventing the formation and clouding of aggregates are as follows. Was not known. Disclosure of the invention

 Therefore, the present invention provides a novel method for effectively suppressing the formation of aggregates or clouding in the ultrafiltration of an antibody-containing solution, and a method which is stabilized against stress by this method. It is an object of the present invention to provide an antibody-containing composition.

 The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, by adding a certain surfactant to the antibody-containing solution, the stress caused by ultrafiltration was alleviated, and the formation of aggregates And clouding can be suppressed.

 Accordingly, the present invention provides a method for suppressing the formation of aggregates or clouding in an antibody solution in ultrafiltration, which comprises adding a surfactant to the antibody-containing solution. The antibody is preferably a monoclonal antibody, and more preferably an anti-IL16 receptor antibody.

 The present invention also provides an antibody-containing composition stabilized against physical stress, wherein the composition contains 0.000025 to 0.5% by weight of a surfactant. The composition is preferably substantially free of salts. The antibody is preferably a monoclonal antibody, and more preferably an anti-IL16 receptor antibody.

 As the surfactant used in the above method and composition, polysorbate and pull-mouth nick are preferable, and polysorbate 20 and polysorbate 80 are particularly preferable. The concentration of the surfactant in the antibody-containing solution is preferably 0.0025 to 0.5% by weight. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a schematic diagram of an apparatus used in an experiment of the present invention.

FIG. 2 is a graph showing the effect of the pressure ratio and the effect of adding polysorbate 80 when an experiment was performed using the apparatus of FIG. FIG. 3 is a graph showing the effect of polysorbate 80 concentration on the suppression of aggregate formation in antibody-containing solutions.

 FIG. 4 is a graph showing the effect of the concentration of polysorbate 80 on the suppression of aggregate formation in an antibody-containing solution.

 FIG. 5 is a graph showing the effect of NaCl on the formation of aggregates in a solution containing antibodies. Embodiment of the Invention

 Although the invention is applicable to polyclonal antibodies, it is particularly preferred to apply to antibodies consisting of a single molecular species. Antibodies comprising a single molecular species include, for example, monoclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, etc., or fragments of these various forms of antibodies. . In particular, chimeric antibodies, humanized antibodies, and single-chain antibodies are more unstable than natural antibodies, and it is preferable to use the method of the present invention.

 Monoclonal antibodies are used, for example, for cell fusion between antibody-producing cells obtained by immunizing animals such as mice, rats, mice, and sheep with antigens, and perpetual cultured cells such as myeloma cells. Is an antibody produced by a hybridoma obtained by the method described above, and a monoclonal antibody against a specific antigen can be produced by a well-known method.

 Human antibodies can be produced by known techniques using transgenic animals.

 A chimeric antibody is composed of a constant region (monoclonal C region) of a monoclonal antibody produced by a hybridoma derived from one organism and a variable region of a monoclonal antibody produced by a hybridoma derived from another organism.

(V-region) and an artificially constructed antibody. As a chimeric antibody, a monoclonal antibody against any antigen It is important that the V-region of a monoclonal antibody other than a human, such as a mouse, be replaced with the C-region of a human monoclonal antibody, which is advantageous in that it is available. . Any chimeric antibody can be produced by well-known techniques.

 The humanized antibody is derived from the CDRs of the human antibody by the complementarity determining regions (CDRs) in the V-region of a monoclonal antibody derived from a non-human animal generated against the desired immunogen. Which comprises a framework region (FR) in the C- and V- regions of a human antibody and a CDR of an antibody derived from an animal other than a human, wherein Since it has a very small region derived from animals other than humans, it is characterized by very low immunogenicity in humans. Any humanized antibody can be produced by well-known techniques.

 A single-chain antibody is one in which the V-region of the light chain (L-chain) and the V-region of the heavy chain (H-chain) are linked by an appropriate linker to form a single chain. The L chain V region and H chain V region may be derived from the same antibody or from different antibodies. Single-chain antibodies can also be produced by well-known techniques.

The present invention is also applicable to fragments of the above-described various forms of antibodies, for example, Fab, F (ab) _{2, and the} like.

 Chimeric antibodies, humanized antibodies, single-chain antibodies, and the like are produced using host cells by genetic recombination. Examples of the host cell include cultured cells of microorganisms or animals. Examples of the host cell include Escherichia coli, yeast, Bacillus subtilis, CHO cells, COS cells, BHK cells, and human-derived cells.

In the present invention, a humanized PM-1 antibody that is an antibody against IL-16 receptor is used as a specific example of the antibody to which the present invention is applied, but the antibody to which the present invention can be applied is not limited thereto. Say not Not even. The humanized PM-1 antibody is a humanized version of the PM_1 antibody, and the PM-1 antibody is a monoclonal antibody against the antigen IL-6 receptor.

 As the surfactant used in the present invention, any surfactant can be used as long as it can stabilize the antibody-containing solution against stress and can prevent the generation of aggregates or clouding in ultrafiltration. Pluronics and the like are mentioned, but polysorbate is preferred, and polysorbate 20 and polysorbate 80 are particularly preferred. The addition concentration is preferably from 0.0025 ° to 0.5% by weight based on the solution.

 Polysorbate is polyoxyethylene ether obtained by esterifying a part of the hydroxyl groups of anhydrous sorbitol with a fatty acid. Polysorbate 20 has lauric acid as its fatty acid, and polysorbate 80 has oleic acid as its fatty acid.

 The antibody-containing solution of the present invention may contain a known buffer such as a phosphate buffer, an acetate buffer, a citrate buffer, and the like. Is preferred.

 The formation or clouding of aggregates in the antibody-containing solution can be measured by any method for measuring them.In the present invention, as a specific example, the absorbance at 340 nm is measured for the sample to be measured before and after filtration. And the difference is defined as the turbidity. In the present invention, the effect of stabilizing stress or the effect of suppressing the formation of aggregates or clouding can be expressed, for example, using the “slope of turbidity” described below as an index.

That is, for example, an ultrafiltration and concentration apparatus as shown in FIG. 1 is used. First, an antibody-containing solution with or without the surfactant to be tested is added to a container 1 indicated by 1 in the figure (control), and the solution is transported to the ultrafilter 3 by the pump 2. I do. In ultrafilter 3, the antibody The solvent constituting the containing solution is subjected to ultrafiltration through the ultrafiltration membrane and discharged from the discharge path 4, and the concentrated antibody-containing solution is returned to the container 1 through the tube 5. By repeating this circulation, the antibody concentration of the solution in the container or the tube 5 increases.

 If the stress caused by the ultrafiltration treatment does not affect the antibody-containing solution at all, that is, if the formation of aggregates or opacity does not occur even by the ultrafiltration treatment, the solution in the vessel 1 or the tube 5 is The antibody concentration increases over time, but the turbidity, ie, the absorbance at 340 nm, does not increase. Therefore, a sample is taken from container 1 with time, the antibody concentration and turbidity are measured, and the antibody concentration is plotted on the horizontal axis and the turbidity is plotted on the vertical axis. The regression line is parallel to the horizontal axis, and the “turbidity slope” is zero.

 On the other hand, when stress is applied to the antibody-containing solution by ultrafiltration and aggregates or turbidity occur, the antibody concentration increases with time and the turbidity also increases. Therefore, in the above graph, the obtained regression line shows a constant “slope” (slope of turbidity). Then, the more susceptible to aggregation and cloudiness in the antibody-containing solution by the ultrafiltration treatment, the greater the “turbidity gradient”. Therefore, the “slope of turbidity” of the antibody-containing solution to which the test surfactant was added and the antibody-containing solution to which no surfactant was added was measured and compared to determine the anti-stress of the test surfactant. The effect, that is, the effect of suppressing aggregate formation or clouding can be measured.

 In addition, various concentrations of sodium chloride were added to the antibody-containing solution, and the `` turbidity gradient '' was measured in the above test.The turbidity gradient increased as the salt concentration increased. It became clear that a lower salt concentration was better to combat stress in filtration.

In carrying out the method of the present invention, prior to ultrafiltration, What is necessary is just to add a predetermined amount of a predetermined surfactant to the solution.

 The present invention also relates to an antibody-containing composition that is stabilized against stress by ultrafiltration by containing a surfactant. This composition is obtained as a result of performing the method of the present invention. The stabilized composition of the present invention may also be a composition solidified by conventional drying means such as freeze drying and vacuum drying. In the solidified stabilized composition, the above-mentioned antibody content means a concentration in a liquid state before solidification. The stress-stabilized antibody-containing composition of the present invention may also be a non-ultrafiltered antibody-containing solution to which a predetermined amount of a surfactant has been added, or a dried product thereof. These compositions exhibit a stabilizing effect during subsequent ultrafiltration. Example

 Next, the present invention will be described more specifically with reference to examples.

 Example 1

 As an example of the apparatus schematically shown in FIG. 1, a Labscale TFF System manufactured by Millipore was used, and Pell icon Biomax 5 manufactured by Millipore was used as an ultrafiltration membrane. A humanized PM-1 antibody was used as the antibody, and the concentration of the antibody solution was measured by a spectrophotometer at 340ηιη. The concentration of the humanized PM_1 antibody was measured by gel filtration.

Approximately 1 _{mg /} mL of humanized PM_1 antibody, sodium phosphate (buffer) 15 mM (ρΗ6.5) and NaCl 120 mM as antibody solution, and predetermined amounts depending on experimental purposes A solution containing polysorbate 80 was prepared and ultrafiltration was continued until the concentration of the humanized PM-11 antibody was concentrated to about 3 to 4 mg / mL.

The stress of ultrafiltration is based on the theory of rheology. Expected to be a function. It was also confirmed that the ultrafiltration rate was a function of the pressure ratio before and after the ultrafiltration membrane. Therefore, the “slope of turbidity” at various pressure ratios was measured for the antibody solution containing 0.005% of polysorbate 80 and the antibody solution containing no polysorbate.

The “slope of turbidity” is obtained by measuring the turbidity in a sample collected over time and the concentration of humanized PM-1 antibody, and taking the value of the absorbance measured at 340 nm as turbidity. as the concentration of preparative reduction PM- 1 antibodies using the value of "m _gm L" human-type horizontal axis PM- 1 antibody concentration [MGZ mL] and the vertical axis taking absorbance measurements This is a numerical value obtained by plotting and calculating the slope of the regression line obtained.

 Figure 2 shows the results of the experiment. As is clear from this figure, the stress due to ultrafiltration, that is, the formation of aggregates or clouding increases with an increase in the pressure ratio (ie, an increase in the ultrafiltration rate). It is suppressed by addition.

 Example 2 Effect of Concentration of Polysorbate 80

 Using the apparatus and method described in Example 1, but using a pressure ratio of 3 and 1.5 and adding polysorbate 80 at 0%, 0.001% and 0.005% concentrations, Degree slope ”was calculated. The results are shown in Figure 3.

 As in the case of Example 1, the stress increased according to the magnitude of the pressure ratio. Also, at all pressure ratios, when 0.001% or more of polysorbate 80 was added, the “turbidity gradient” was reduced, and the generation of aggregates was suppressed.

 Example 3. Effect of polysorbate concentration

The same apparatus and the same method as described in Example 1 were used, but the pressure ratio was fixed at 3, and the concentration of polysorbate 80 was 0%, 0.00025%, 0.0005%, and 0.005%. 0010%, 0.002%, and 0.004% The slope was measured. Fig. 4 shows the results. It was found that a sufficient anti-stress effect, that is, an effect of suppressing aggregate formation, was obtained when the concentration of polysorbate 80 was 0.00025%.

 Example 4. Effect of sodium chloride

 The same apparatus and method as described in Example 1 was used, but the pressure ratio was fixed at about 1.5, no polysorbate was added, no NaCl concentration was added, 120 mM and 100 mM. Ultrafiltration was performed from the initial concentration of the humanized PM_1 antibody concentration of about 1 mg / mL to the concentration of about 40 mg / mL. Fig. 5 shows the results. In the absence of NaCl, the “turbidity slope” was the lowest, indicating that NaCl promoted the formation of aggregates in the antibody solution.

 Reference Example 1. Preparation of human IL-16 receptor antibody PM-1

 Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, Inc.) in which the anti-IL-16R antibody MT18 prepared by the method of Hirata et al. (J. Immunol., 143: 2900-2906, 1989) was activated by CNBr. NJ) and purified according to the attached procedure, and purified IL-6R (Yamasaki et al., Science 241: 825-828, 1988).

 That is, the human myeloma cell line U266 was transformed with ImM p-paraaminophenol 2 containing 1% digitonin (Wako Chemicals), 10 mM triethanolamine (pH 7.8) and 0.15 M NaCl. The protein was solubilized with dehydrochloride (manufactured by Wako Chemicals) (digitonin buffer), and mixed with MT18 antibody bound to Sepharose 4B beads. Thereafter, the beads were washed six times with digitonin buffer to obtain partially purified IL-16R used for immunization.

BALB / c mice were immunized four times every 10 days with the above partially purified IL-6R obtained from 3 × 10 ⁹ U266 cells, and then a hybridoma was prepared by a conventional method. The hybridoma culture supernatant from the growth positive The binding activity to IL-16R was examined by the method. 5 × 10 ⁷ U266 cells were labeled with 35 S-methionine (2.5 mCi) and solubilized with the above digitonin buffer.

The solubilized U266 cells were mixed with 0.04 ml of MT18 antibody conjugated to Sepharose 4B beads, then washed six times with digitonin buffer, and washed with 0.25 ml of digitonin buffer (pH 3.4). Ri drained the ³⁵ S- Mechionin labeled IL- 6R, and neutralized with 1 M Tris of 0.025 ml (pH 7.4). 0.05 ml of the hybridoma culture supernatant was mixed with 0.01 ml of Protein G Sepharose (Pharmacia).

After washing, the Sepharose was both Inkyubeto with ³⁵ S-labeled IL-6R solution 0.005ml prepared above. The immunoprecipitated substances were analyzed by SDS-PAGE, and the hybridoma culture supernatant that reacted with IL-6R was examined. As a result, a reaction-positive hybridoma clone PM_1 was established. The IL-6R antibody PM-1 produced from the hybridoma PM-1 has an IgGl _K- type subtype.

The activity of the antibody produced by hybridoma PM-1 to inhibit the binding of IL-16R to human IL-16R was examined using the human myeloma cell line U266. Fire Efforts replaceable IL one 6R prepared Ri by E. coli (Hirano et al., Immunol Lett, 17:.. 41, 1988), Bonore Bok emissions - Nono centers reagent (New England Nuclear, Boston, MA ) Ri by the ¹²⁵ I-labeled (Taga et al., J. Exp. Med. 166: 967, 1987).

4 × 10 ⁵ U266 cells were cultured for 1 hour at room temperature in the presence of 100-fold excess of unlabeled IL-6R at 70% (V / V) of the culture supernatant of Hypridoma PM-1. The cells were cultured with 14000 CPM of ¹²⁵ I-labeled IL-16R. 701 samples were overlaid on 300 μl FCS in a 400 μl microfuge polyethylene tube, and after centrifugation the radioactivity on the cells was measured. As a result, the antibody produced by Hypri-Doma ΡΜ-1 It was found to inhibit the binding of IL-6R to IL-6R. Reference Example 2. Preparation of humanized PM-1 antibody

 The humanized PM_1 antibody was obtained by the method described in International Patent Application Publication No. W092-19759. Total RNA was prepared from the hybridoma PM-1 prepared in Reference Example 1 by a conventional method, and single-stranded cDNA was synthesized therefrom. The DNA in the V region of mouse PM-1 was amplified by the polymerase chain reaction (PCR) method. The primers used in the PCR method were those described in ST Jones et al., Bio / Technology, 9, 88, 1991.

 The DNA fragment amplified by the PCR method was purified to obtain a DNA fragment containing a gene encoding a mouse kappa type L chain V region and a DNA fragment containing a gene encoding a mouse gamma type H chain variable region. These DNA fragments were ligated to a plasmid PUC19 and introduced into E. coli DH5α recombinant cells to obtain E. coli transformants. The above plasmid was obtained from this transformant, the nucleotide sequence of the cDNA coding region in the plasmid was determined according to a conventional method, and the complementarity determining region (CDR) of each V region was determined.

 In order to prepare vectors expressing the chimera PM_1 antibody, cDNAs encoding the mouse PM-1 KL light chain and H chain V region, respectively, were inserted into an HCMV expression vector. In order to prepare a humanized PM_1 antibody, the mouse PM-1 V region CDR was transplanted into the human antibody by the CDR transplantation method. The amino acid in the framework (FR) region of the variable region of the antibody was replaced so that the CDR of the humanized antibody formed an appropriate antigen-binding site.

In order to express the L-chain and H-chain genes of the humanized PM-1 antibody thus prepared in mammalian cells, each vector containing the human hormone factor la (HEF-1a) promoter was used. Each vector was introduced and a vector expressing the humanized PM-1 antibody L chain and H chain was prepared. Simultaneously import these two expression vectors into CH0 cells As a result, a cell line producing a humanized PM-1 antibody was established. The binding ability of the obtained humanized antibody to human IL-6R was confirmed by ELISA. In addition, hPM-1 inhibited binding of human IL-6 to human IL-6R, as did mouse and chimeric antibodies.

Claims

The scope of the claims 1. A method for suppressing the formation of aggregates or clouding in an antibody-containing solution in ultrafiltration, which comprises adding a surfactant to the antibody-containing solution.  2. The method of claim 1, wherein the surfactant is a polysorbate.  3. The method according to claim 2, wherein the surfactant is polysorbate 20 or polysorbate 80.  4. The method according to any one of claims 1 to 3, wherein the concentration of the surfactant added to the antibody-containing solution is 0.00025 to 0.5% by weight based on the solution.  5. The method according to any one of claims 1 to 4, wherein the antibody-containing solution is substantially free of salts.  6. The method according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.  7. The method according to any one of claims 1 to 5, wherein the antibody is an antibody produced in CH0 cells.  8. The method according to any one of claims 1 to 5, wherein the antibody is a chimeric antibody or a humanized antibody.  9. The method according to any one of claims 1 to 8, wherein the antibody is an anti-IL-6 receptor antibody.  10. An antibody composition stabilized against shear stress, wherein the composition comprises 0.0005 to 0.5% by weight of a surfactant. 11. 0.00025-0.5 weight of surfactant in antibody-containing composition stabilized against shear stress. /. Containing, and 11. The composition according to claim 10, wherein the composition is substantially free of salts.  12. The antibody-containing composition according to claim 10, wherein the surfactant is polysorbate.  13. The antibody-containing composition according to claim 12, wherein the polysorbate is polysorbate 20 or polysorbate 80.  14. The antibody-containing composition according to any one of claims 10 to 13, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.  15. The method according to any one of claims 10 to 14, wherein the antibody is an anti-IL-16 receptor antibody.