Improved process for affinity chromatography

Abstract

"Improved Process for Affinity Chromatography" A process for purifying antibodies or fusion proteins by affinity chromatography, wherein in the neutralization step, elution is performed at a high salt concentration that reduces turbidity in the protein mixture. The present invention provides an improved process for purifying antibodies by affinity chromatography using high-salt elution.

Description

Technical Field

[0001] The present invention provides a composition of antibodies obtained from affinity chromatography that can provide low turbidity during virus inactivation and neutralization. The present invention provides an improved process for purifying antibodies by affinity chromatography using high-salt elution.

Background Art

[0002] Viruses are potential contaminants in the manufacturing process of drugs, especially when biological drugs are derived from mammalian cell cultures. Sources of virus contaminants can be the medium used for cell culture or the cell line that produces the biological drug of interest.

[0003] Monoclonal antibodies are widely purified by protein A affinity chromatography. Affinity chromatography has several advantages as it is a simple, rapid, and selective procedure for capturing target proteins. Due to its selectivity, an affinity purification step is generally introduced early in the purification chain. This can reduce the number of consecutive unit operations. Since a cost-effective manufacturing process is required, optimization of downstream purification including the affinity step is necessary. In addition to the efficient removal of process-related impurities such as host cell proteins and DNA, protein A has also been claimed to remove viruses. Protein A is eluted at acidic pH, and chemical inactivation of enveloped viruses by denaturation of envelope proteins is typically performed. Usually, this inactivation step is carried out at pH 3.0 - 4.0. It has been reported that the higher the pH and the lower the temperature, the slower the inactivation reaction rate.

[0004] However, when subjecting an antibody composition to virus inactivation treatment, during the elution of monoclonal antibodies (mAbs) at acidic pH in protein A chromatography, turbid elution pools and high column backpressure are common.

[0005] Filtration is a crucial unit operation used for primary and secondary clarification during the production of biological preparations from mammalian cell lines. However, continuous production processes require consistent use of filtration over long periods, and unpredictable fluctuations in the attributes of the feed stream can occur, which is a challenge currently faced in this field. Furthermore, increased turbidity ultimately leads to the use of multiple filters or filters with large filtration areas, directly impacting process costs and operating time. In addition, deep filtration is used to remove precipitates and turbidity after neutralization of low-pH virus inactivation products. Furthermore, this process can be improved by applying positively charged filters to further remove host cell proteins (HCPs) and DNA. Advocating for adsorbent deep filtration as a virus removal step may allow for further enhancement of the mAb bioprocess. This invention solves this problem by providing a low-turbidity protein mixture during or after neutralization that requires a small filtration area. Deep filtration is also commonly used to remove precipitates and turbidity after neutralization of intermediates of low-pH virus inactivation products.

[0006] Therefore, there remains a need in this field to develop processes for removing or reducing turbidity and precipitates during virus inactivation. [Overview of the project] [Problems that the invention aims to solve]

[0007] In one embodiment, the present invention provides an improved purification process for antibodies or fragments thereof by using affinity chromatography in which elution is performed at a high salt concentration, which provides lower turbidity or precipitation in the eluted composition during virus inactivation and neutralization compared to elution performed at a low salt concentration.

[0008] In one embodiment of this invention, the present invention provides a high salt concentration during elution, selected from over 100 mM, over 110 mM, over 125 mM, over 130 mM, over 140 mM, over 150 mM, over 160 mM, over 170 mM, over 180 mM, over 190 mM, over 195 mM, and about 200 mM.

[0009] In one embodiment of this invention, the present invention provides low turbidity or precipitation in an eluted composition selected from about 10 NTU, about 20 NTU, about 30 NTU, about 35 NTU, about 36.1 NTU, about 40 NTU, about 42.5 NTU, about 50 NTU, about 60 NTU, about 70 NTU, about 80 NTU, about 90 NTU, and about 100 NTU.

[0010] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 100 mM to approximately 200 mM at an appropriate acidic pH; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process having a turbidity of less than 100 NTU when measured with a standard turbidimeter.

[0011] In one embodiment, the elution pH is selected from pH 3 to pH 3.5.

[0012] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 125 mM at an appropriate acidic pH of 3.5 ± 0.1; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process that results in a turbidity of 103, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM, as measured with a standard turbidimeter.

[0013] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 200 mM at an appropriate acidic pH of 3.5 ± 0.1; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process that, when measured with a standard turbidimeter, has a turbidity of 36.1, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM.

[0014] In one embodiment, the present invention is a process for purifying a protein mixture, a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with a suitable buffer; d. Elute the protein mixture concentrated with the antibody of interest with a suitable buffer having a concentration of about 200 mM at a suitable acidic pH of 3.0 ± 0.1; e. Inactivate and neutralize the virus in the eluted protein mixture, and the neutralized protein mixture has a turbidity of 42.5, which is lower than the protein mixture eluted with a buffer having a concentration of less than 100 mM when measured with a standard turbidimeter.

[0015] In one embodiment, the present invention is a process for purifying a protein mixture, the process comprising: a. Loading a protein mixture of an antibody or a fragment thereof and impurities onto an affinity column using a suitable buffer; b. Washing the affinity column with a suitable buffer; c. Optionally, perform another wash with a suitable buffer; d. Eluting the protein mixture concentrated with the antibody of interest with a suitable buffer having a concentration of about 125 mM; e. Inactivate and neutralize the virus in the eluted protein mixture, and the protein mixture provides a process having a turbidity of less than 50 NTU when measured with a standard turbidimeter.

[0016] In one embodiment, the present invention is a process for purifying a protein mixture, the process comprising: a. Loading a protein mixture of an antibody or a fragment thereof and impurities onto an affinity column using a suitable buffer; b. Washing the affinity column with a suitable buffer; c. Optionally, perform another wash with a suitable buffer; d. Eluting the protein mixture concentrated with the antibody of interest with a suitable buffer having a concentration of about 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture; and providing a process comprising the protein mixture having a turbidity of less than 50 NTU when measured with a standard turbidimeter.

[0017] In one embodiment, the present invention is a process for purifying a protein mixture, the process comprising: a. Loading a protein mixture of omalizumab or a fragment thereof and impurities onto an affinity column using a suitable buffer; b. Washing the affinity column with a suitable buffer; c. Optionally, washing again with a suitable buffer; d. Eluting the protein mixture concentrated with omalizumab with a suitable buffer having a concentration of about 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture; and providing a process comprising the protein mixture having a turbidity of less than 20 NTU when measured with a standard turbidimeter.

[0018] In one embodiment, the present invention is a process for purifying a protein mixture, the process comprising: a. Loading a protein mixture of an antibody or a fragment thereof and impurities onto an affinity column using a suitable buffer; b. Washing the affinity column with a suitable buffer; c. Optionally, washing again with a suitable buffer; d. Eluting the protein mixture concentrated with the target antibody with a suitable buffer having a concentration of about 125 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture; and providing a process comprising the protein mixture having a turbidity of less than 100 NTU when measured with a standard turbidimeter.

Brief Description of the Drawings

[0019] [Figure 1] Figure 1 shows the complete chromatogram. [Modes for carrying out the invention]

[0020] definition The term "antibody" includes immunoglobulin molecules composed of four polypeptide chains, namely two heavy chains (H) and two light chains (L), linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain more conserved regions called framework regions (FRs). VH and VL are each composed of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus.

[0021] In this specification, the expression “reduction / inactivation of virus” is intended to mean a reduction in the number of virus particles in a particular sample ("reduction") and a reduction in the activity of virus particles in a particular sample, for example, a reduction in their infectivity or replication ability ("inactivation"). Such reduction in the number and / or activity of virus particles may be on the order of about 1% to about 99%, preferably about 20% to about 99%, more preferably about 30% to about 99%, more preferably about 40% to about 99%, even more preferably about 50% to about 99%, even more preferably about 60% to about 99%, even more preferably about 70% to about 99%, even more preferably about 80% to 99%, and even more preferably about 90% to about 99%.

[0022] As used herein, “comprises” or “comprising” does not exclude other elements or processes. For the purposes of the present invention, the term “consisting of” is considered to be any embodiment of the term “comprising of.” Hereinafter, where a group is defined to include at least a certain number of embodiments, this is also understood to disclose a group that may consist only of these embodiments.

[0023] Throughout the specification and the attached claims, the singular forms "a," "an," and "the" include references to the plural, unless the context clearly indicates that they are not plural.

[0024] In this specification, the term "about" is intended to mean a range of about 10-20% greater or less than the reference value. In certain circumstances, a person skilled in the art will recognize that, depending on the nature of the reference value, the term "about" may mean a value greater or less than a 10-20% deviation from that value.

[0025] Omalizumab (Xolair®) is a recombinant DNA-derived humanized IgG1K monoclonal antibody that selectively binds to human immunoglobulin (IgE). The molecular weight of the antibody is approximately 149 kD. Xolair® is produced by suspension culture of Chinese hamster ovary cells in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detected in the final product. Xolair® is a sterile, white, preservative-free, lyophilized powder contained in a single-use vial, reconstituted with sterile water for injection (SWFI) USP, and administered as a subcutaneous (SC) injection.

[0026] Turbidity is generally the cloudiness or haze of a liquid caused by numerous individual particles that are invisible to the naked eye, similar to smoke in the air. The property of particles to scatter focused light rays is now considered a more meaningful measure of turbidity in water.

[0027] Turbidity measured using this method is determined using an instrument called a turbidimeter, which has a detector set up next to the light beam. When there are many small particles scattering the source beam, more light reaches the detector than when there are few. The unit of turbidity measured by a calibrated turbidimeter is called the turbidimetric turbidity unit (NTU).

[0028] In protein purification, turbidity plays a crucial role. Highly turbid protein solutions contain more insoluble particles, which can be aggregates or other impurities, negatively impacting the purity and yield of the protein mixture. High turbidity can also cause cassettes and columns to clog and break. Therefore, high turbidity indicates low purity and high impurity concentrations, and vice versa.

[0029] This process significantly reduces the turbidity of the protein A eluate in response to the protein A input.

[0030] In one embodiment, the present invention provides an improved purification process for antibodies or fragments thereof by using affinity chromatography in which elution is performed at a high salt concentration, which provides lower turbidity or precipitation in the eluted composition compared to elution performed at a low salt concentration.

[0031] In one embodiment of this invention, the present invention provides a high salt concentration during elution, selected from over 100 mM, over 110 mM, over 125 mM, over 130 mM, over 140 mM, over 150 mM, over 160 mM, over 170 mM, over 180 mM, over 190 mM, over 195 mM, and about 200 mM.

[0032] In one embodiment of this invention, the present invention provides low turbidity or precipitation in an eluted composition selected from about 10 NTU, about 20 NTU, about 30 NTU, about 35 NTU, about 36.1 NTU, about 40 NTU, about 42.5 NTU, about 50 NTU, about 60 NTU, about 70 NTU, about 80 NTU, about 90 NTU, and about 100 NTU.

[0033] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 100 mM to approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The process provides a protein mixture having lower turbidity than the protein mixture eluted with a buffer having a concentration of less than 100 mM, as measured with a standard turbidimeter.

[0034] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 100 mM to approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture comprises a process that, when measured with a standard turbidimeter, has a turbidity of less than 100 NTU.

[0035] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 125 mM at an appropriate acidic pH of 3.5 ± 0.1; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process that results in a turbidity of 103, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM, as measured with a standard turbidimeter.

[0036] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 200 mM at an appropriate acidic pH of 3.5 ± 0.1; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process that, when measured with a standard turbidimeter, has a turbidity of 36.1, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM.

[0037] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 200 mM at an appropriate acidic pH of 3.0 ± 0.1; e. Inactivating and neutralizing the virus in the eluted protein mixture, The neutralized protein mixture, containing the above, provides a process that, when measured with a standard turbidimeter, has a turbidity of 42.5, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM.

[0038] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 125 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture comprises a process that, when measured with a standard turbidimeter, has a turbidity of less than 100 NTU.

[0039] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture contains the following, and when measured with a standard turbidimeter, it has a turbidity of less than 100 NTU.

[0040] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with the antibody of the target in a suitable buffer having a concentration of approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture comprises a process that provides a turbidity of less than 50 NTU when measured with a standard turbidimeter.

[0041] In another embodiment, using a high salt concentration during the elution of protein A improves the filtration capacity, allowing the neutralized protein mixture to be filtered with a small filter area.

[0042] In another embodiment, using a high salt concentration during the elution of protein A improves the filtration capacity by 1, 1.5, or 2 times.

[0043] In certain embodiments, the antibody is selected from IgG1, IgG2, IgG3, and IgG4 antibodies or fragments thereof, and fusion proteins. In one embodiment, the IgG1 antibody or fusion protein has an isoelectric point of 6 to 9.

[0044] The IgG1 antibody or fusion protein mentioned above is etanercept, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab, omalizumab, cetuximab, bevacizumab, natalizumab, eculizumab, certolizumab pegol, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, trastuzumab emtansine, lacryma The following drugs are selected: cibakumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, nivolumab, pembrolizumab, darucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, sarilumab, ocrelizumab, tildrakizumab, romosozumab, brolucizumab, and crizanlizumab.

[0045] In a preferred embodiment, the antibody is an anti-IgE antibody. In a specific embodiment, the anti-IgE antibody is omalizumab.

[0046] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of omalizumab or its fragments and impurities into an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with omalizumab in a suitable buffer having a concentration of approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture comprises a process having a turbidity of less than 20 NTU when measured with a standard turbidimeter.

[0047] In one embodiment, the present invention is a process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of omalizumab or its fragments and impurities into an affinity column using an appropriate buffer; b. Wash the affinity column with an appropriate buffer; c. Optionally, perform another wash with an appropriate buffer; d. Elute the protein mixture concentrated with omalizumab in a suitable buffer having a concentration of approximately 200 mM; e. Inactivating and neutralizing the virus in the eluted protein mixture, The protein mixture comprises a process that, when measured with a standard turbidimeter, has a turbidity of less than 10 NTU.

[0048] In one embodiment, the protein mixture eluted with a buffer having a concentration of 100 mM to 250 mM has lower turbidity than the protein mixture eluted with a buffer having a concentration of less than 100 mM.

[0049] In one embodiment, the protein mixture eluted with a buffer having a concentration of 200 mM has lower turbidity than the protein mixture eluted with a buffer having a concentration of 30 mM.

[0050] In one embodiment, the protein mixture eluted with a buffer having a concentration of 100 mM to 125 mM has lower turbidity than the protein mixture eluted with a buffer having a concentration of less than 30 mM.

[0051] In one embodiment, the affinity chromatography is selected from protein A or protein G. In one embodiment, the affinity chromatography resin is selected from Mabselect, Mabselect SuRe, Mabselect SuRe Lx, Prosep Ultra Plus, and Eshmuno A. In a preferred embodiment, the affinity chromatography resin is Mabselect Sure Lx.

[0052] In one embodiment, the equilibration buffer, load buffer, or wash buffer is selected from sodium phosphate, tris-HCl, tris-acetate, HEPES, and glycine-NaOH. In a preferred embodiment, the load buffer is tris-acetate.

[0053] In certain embodiments, the equilibration buffer, load buffer, or wash buffer is used in combination with a salt. In certain embodiments, the salt is selected from sodium chloride, potassium chloride, arginine chloride, calcium chloride, and urea. In preferred embodiments, the salt is sodium chloride.

[0054] In one embodiment, the equilibration buffer has a concentration range of about 5 mM to about 40 mM. In a particular embodiment, the equilibration buffer has a concentration range of about 10 mM to about 25 mM. In a preferred embodiment, the concentration of the equilibration buffer is about 20 mM.

[0055] In certain embodiments, the equilibration buffer, or load buffer, or wash buffer optionally contains a salt selected from about 50 mM to about 400 mM. In one embodiment, the equilibration buffer contains a salt buffer concentration selected from about 100 mM to about 200 mM. In one embodiment, the concentration of the equilibration buffer is about 150 mM. In another embodiment, the concentration of the equilibration buffer is about 100 mM.

[0056] In one embodiment, the equilibrating buffer, load buffer, or wash buffer has a conductivity range of approximately 10 mS / cm to approximately 20 mS / cm. In another embodiment, the conductivity of the equilibrating buffer, load buffer, or wash buffer is approximately 15.0 mS / cm to 20.0 mS / cm. In yet another embodiment, the conductivity of the equilibrating buffer, load buffer, or wash buffer is approximately 10.0 mS / cm to 13.0 mS / cm.

[0057] In one embodiment, the pH of the equilibration buffer, load buffer, or wash buffer is selected from about 6.5 to about 7.5. In a preferred embodiment, the pH of the equilibration buffer is about 7.0.

[0058] In one embodiment, the load buffer has a concentration range of about 5 mM to about 40 mM. In another embodiment, the load buffer has a concentration range of about 10 mM to about 30 mM. In a preferred embodiment, the concentration of the load buffer is about 20 mM.

[0059] In a particular embodiment, the affinity chromatography has at least one wash buffer. In another embodiment, the affinity chromatography has three wash buffers.

[0060] In one embodiment, the first washing buffer has a concentration range of about 5 mM to about 40 mM. In a particular embodiment, the first washing buffer has a concentration range of about 10 mM to about 25 mM. In a preferred embodiment, the concentration of the first washing buffer is about 20 mM.

[0061] In one embodiment, the second washing buffer is selected from sodium phosphate, tris-HCl, tris-acetate, HEPES, and glycine-NaOH.

[0062] In certain embodiments, the second washing buffer is used in combination with a salt.

[0063] In certain embodiments, the salt is selected from sodium chloride, potassium chloride, arginine chloride, calcium chloride, and urea. In preferred embodiments, the salt is sodium chloride.

[0064] In one embodiment, the second washing buffer has a concentration range of about 5 mM to about 40 mM. In a particular embodiment, the second washing buffer has a concentration range of about 10 mM to about 25 mM. In a preferred embodiment, the concentration of the second washing buffer is about 20 mM.

[0065] In one embodiment, the second washing buffer has a salt buffer concentration range of about 0.5 M to about 1.5 M. In a preferred embodiment, the concentration of the second washing buffer is about 1.0 M.

[0066] In one embodiment, the second cleaning buffer has a conductivity range of about 70 mS / cm to about 120 mS / cm. In another embodiment, the second cleaning buffer has a conductivity range of about 80 mS / cm to about 100 mS / cm. In a preferred embodiment, the conductivity of the second cleaning buffer is about 90 mS / cm.

[0067] In one embodiment, the pH of the second washing buffer is selected from about 6.5 to about 7.5. In a preferred embodiment, the pH of the second washing buffer is about 7.0.

[0068] In one embodiment, the second washing buffer further comprises a surfactant selected from polysorbate 20, polysorbate 80, and Triton X-100. In one embodiment, the preferred surfactant is polysorbate 20.

[0069] In one embodiment, the proportion of surfactant in the second washing buffer is approximately 0.1% to approximately 1%.

[0070] In one embodiment, the third washing buffer has a concentration range of about 5 mM to about 40 mM. In a particular embodiment, the third washing buffer has a concentration range of about 10 mM to about 30 mM. In a preferred embodiment, the concentration of the third washing buffer is about 20 mM.

[0071] In one embodiment, the third cleaning buffer has a conductivity range of about 0.5 mS / cm to about 2.5 mS / cm. In a preferred embodiment, the conductivity of the third cleaning buffer is about 1 mS / cm.

[0072] In one embodiment, the pH of the third washing buffer is selected from about 5 to about 6. In a preferred embodiment, the pH of the third washing buffer is about 5.5.

[0073] In one embodiment, the elution buffer is selected from acetic acid, phosphoric acid, and HCl. In a preferred embodiment, the elution buffer is acetic acid.

[0074] In one embodiment, the elution buffer has a concentration range selected from about 100 mM to about 250 mM. In a preferred embodiment, the elution buffer has a concentration range of about 200 mM.

[0075] In one embodiment, the elution buffer has a conductivity range selected from about 0.2 mS / cm to about 0.7 mS / cm. In one embodiment, the elution buffer has a conductivity range selected from about 0.5 mS / cm to about 0.6 mS / cm. In one embodiment, the elution buffer has a conductivity range selected from about 0.2 mS / cm to about 0.3 mS / cm.

[0076] In one embodiment, the pH of the elution buffer is selected from 2.5 to about 3.5. In a preferred embodiment, the pH of the elution buffer is about 3.0.

[0077] In certain embodiments, elution is carried out with a linear gradient. In certain embodiments, elution is carried out with a stepped gradient.

[0078] In one embodiment, the recovery of the elution peak is started at a value of approximately 2.5 AU / cm above the normal range and ended at a value of approximately 2.5 AU / cm below the normal range.

[0079] In one embodiment, the recovery of the elution peak is started at an upper value of approximately 0.25 AU / cm and ended at a lower value of approximately 0.25 AU / cm.

[0080] In one embodiment, the present invention provides an antibody composition having a turbidity selected from less than about 100 NTU, less than about 50 NTU, less than about 30 NTU, and less than about 10 NTU, obtained from affinity chromatography in which the elution buffer has a concentration of about 200 mM.

[0081] In another embodiment, the present invention provides a purification process for antibodies or fragments thereof using affinity chromatography that performs elution at low salt concentrations.

[0082] In another embodiment, the present invention provides a purification process for antibodies or fragments thereof using affinity chromatography that performs elution at a low salt concentration, wherein the turbidity of the eluted protein mixture does not decrease during virus inactivation compared to elution performed at a high salt concentration.

[0083] In one embodiment, the equilibration is performed at approximately 3 CV to approximately 10 CV. In a preferred embodiment, the equilibration is performed at approximately 5 CV. In one embodiment, the equilibration is performed until the conductivity endpoint of the equilibration buffer is achieved.

[0084] In one embodiment, the amount of protein loaded into the column during loading is in the range of approximately 10 g / l to approximately 40 g / l. In another embodiment, the amount of protein loaded into the column during loading is in the range of approximately 10 g / l to approximately 50 g / l.

[0085] In one embodiment, the first wash is performed for at least 1 CV to about 5 CV. In a preferred embodiment, the first wash is performed for 3 CV. In one embodiment, the first wash is performed until the buffer conductivity endpoint is achieved.

[0086] In one embodiment, the second wash is performed for at least 1 CV to about 5 CV. In a preferred embodiment, the second wash is performed for 5 CV. In one embodiment, the second wash is performed until the buffer conductivity endpoint is achieved.

[0087] In one embodiment, the third wash is performed at least 4CV to about 8CV. In a preferred embodiment, the third wash is performed at 7CV. In one embodiment, the third wash is performed until the buffer conductivity endpoint is achieved.

[0088] In one embodiment, the residence time of the protein in the column during protein A purification is in the range of about 2 minutes to about 6 minutes. In a preferred embodiment, the residence time of the protein in the column is about 4 minutes.

[0089] Example 1 - Purification of monoclonal antibodies by protein A chromatography (affinity) using 25 mM acetate buffer and pH 3.5. All chromatography processes were performed using a Cytiva AKTA Pure 150 system. Protein sample concentrations were determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. Mabselect Sure LX resin media was obtained from Cytiva. Vantage columns were obtained from Millipore Corporation. Turbidity was measured using a TN100 portable turbidimeter obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were GMP grade.

[0090] Using Protein A (Mab Select Sure LX, Cytiva) packed into a Vantage VL11×30 Millipore column, monoclonal antibody molecules capable of binding to IgE molecules expressed in Chinese hamster ovary (CHO) cell lines were captured. The residence time was 4 minutes for all phases. After equilibration with Tris-acetate + 150 mM NaCl, pH 6.8~7.2, the clarified product was loaded with resin at ≤40 mg / mL. As shown in Table 1, after loading, the column was washed with equilibration buffer, followed by washing with Wash 2 buffer and Wash 3 buffer, and elution was performed with 25 mM acetate, pH 3.5±0.1 buffer. The affinity chromatography process was operated in binding and elution modes, and the peak was recovered from 500 mAU (2.5 AU / cm) above to 500 mAU (2.5 AU / cm) below. The eluate was further subjected to virus inactivation and neutralization. Turbidity was measured during the protein A elution stage.

[0091] Virus inactivation is performed by adjusting the pH of the protein A eluate to pH 3.5 with 1N HCl and incubating at room temperature for 50 minutes. After 50 minutes of incubation, the sample is neutralized to pH 6.2 with 1M Tris base for 20 minutes. The NTU of the neutralized protein A eluate is measured using a turbidimeter. The neutralized protein A eluate is filtered through a 0.2 μm filter. The experimental design and NTU data for the protein A step are summarized in Table 1 and Table 5, respectively. Table 1: Experimental design of protein A chromatography [Table 1]

[0092] Example 2 - Purification of monoclonal antibodies by protein A chromatography (affinity) using 125 mM acetate buffer, pH 3.5. All chromatography processes were performed using a Cytiva AKTA Pure 150 system. Protein sample concentrations were determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. Mabselect Sure LX resin media were obtained from Cytiva. XK50 / 40 columns were also obtained from Cytiva. Turbidity was measured using a Thermo Scientific TN100 portable turbidimeter. All chemicals were obtained from JTB or Merck Millipore and were GMP grade.

[0093] Monoclonal antibody molecules capable of binding to IgE molecules expressed in Chinese hamster ovary (CHO) cell lines were captured using Protein A (Mab Select Sure LX, Cytiva) packed into an XK50 / 40 column. The residence time was 4 minutes for all phases. After equilibration with Tris-acetate + 150 mM NaCl, pH 6.8-7.2, the clarified product was loaded with resin at a concentration of ≤40 mg / mL. As shown in Table 2, after loading, the column was washed with equilibration buffer, followed by column washing with Wash 2 buffer and Wash 3 buffer, and elution was performed with 125 mM acetate, pH 3.5 ± 0.1 buffer. The affinity chromatography process was operated in binding and elution modes, and the eluate was collected from 500 mAU (2.5 AU / cm) above the peak to 500 mAU (2.5 AU / cm) below the peak. The eluate was further subjected to virus inactivation and neutralization. Turbidity was measured at the Protein A elution stage.

[0094] Virus inactivation is performed by adjusting the pH of the protein A eluate to pH 3.5 with 1N HCl and incubating at room temperature for 50 minutes. After 50 minutes of incubation, the sample is neutralized to pH 6.2 with 1M Tris base for 20 minutes. The NTU of the neutralized protein A eluate is measured using a turbidimeter. The neutralized protein A eluate is filtered through a 0.2 μm filter. The experimental design and NTU data for the protein A step are summarized in Tables 2 and 5, respectively. Table 2: Experimental design of protein A chromatography [Table 2]

[0095] Example 3 - Purification of monoclonal antibodies by protein A chromatography (affinity) using 200 mM acetate buffer and pH 3.5. All chromatography processes were performed using a Cytiva AKTA Pure 150 system. Protein sample concentrations were determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. Mabselect Sure LX resin media was obtained from Cytiva. Vantage columns were obtained from Millipore Corporation. Turbidity was measured using a TN100 portable turbidimeter obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were GMP grade.

[0096] Using a Vantage VL11×30 Millipore column packed with Protein A (Mab Select Sure LX, Cytiva), monoclonal antibody molecules capable of binding to IgE molecules expressed in Chinese hamster ovary (CHO) cell lines were captured. The residence time was 4 minutes for all phases. After equilibration with Tris-acetate + 150 mM NaCl, pH 6.8-7.2, the clarified product was loaded with resin at ≤40 mg / mL. As shown in Table 1, after loading, the column was washed with equilibration buffer, followed by column washing with Wash 2 buffer and Wash 3 buffer, and elution was performed with 200 mM acetate, pH 3.5 ± 0.1 buffer. The affinity chromatography process was operated in binding and elution modes, and the peak was recovered from 500 mAU (2.5 AU / cm) above to 500 mAU (2.5 AU / cm) below. The eluate was further subjected to virus inactivation and neutralization. Turbidity was measured during the protein A elution stage.

[0097] Virus inactivation is performed by adjusting the pH of the protein A eluate to pH 3.5 with 1N HCl and incubating at room temperature for 50 minutes. After 50 minutes of incubation, the sample is neutralized to pH 6.2 with 1M Tris base for 20 minutes. The NTU of the neutralized protein A eluate is measured using a turbidimeter. The neutralized protein A eluate is filtered through a 0.2 μm filter. The experimental design and NTU data for the protein A process are summarized in Tables 3 and 5, respectively. Table 3: Experimental design of protein A chromatography [Table 3]

[0098] Example 4 - Purification of monoclonal antibodies by protein A chromatography (affinity) using 200 mM acetate buffer and pH 3.0. All chromatography processes were performed using a Cytiva AKTA Pure 150 system. Protein sample concentrations were determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. Mabselect Sure LX resin media was obtained from Cytiva. Vantage columns were obtained from Millipore Corporation. Turbidity was measured using a TN100 portable turbidimeter obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were GMP grade.

[0099] Using Protein A (Mab Select Sure LX, Cytiva) packed into a Vantage VL11×30 column, monoclonal antibody molecules capable of binding to IgE molecules expressed in Chinese hamster ovary (CHO) cell lines were captured. The residence time was 4 minutes for all phases. After equilibration with Tris-acetate + 100 mM NaCl, pH 6.8-7.2, the clarified product was loaded with resin at ≤40 mg / mL. As shown in Table 3, after loading, the column was washed with equilibration buffer, followed by column washing with Wash 2 buffer and Wash 3 buffer, and elution was performed with 200 mM acetate, pH 3.0 ± 0.1 buffer. The affinity chromatography process was operated in binding and elution modes, and the peak was recovered from 50 mAU (0.25 AU / cm) above to 50 mAU (0.25 AU / cm) below. The eluate was further subjected to virus inactivation and neutralization. Turbidity was measured during the protein A elution stage.

[0100] Virus inactivation is performed by adjusting the pH of the protein A eluate to 3.5 with 1N acetic acid and incubating at room temperature for 50 minutes. After 50 minutes of incubation, the sample is neutralized to pH 6.2 with 1M Tris base. The NTU of the neutralized protein A eluate is measured using a turbidimeter. The neutralized protein A eluate is filtered through a 0.2 μm filter. The experimental design and NTU data for the protein A step are summarized in Tables 4 and 5, respectively. Table 4: Experimental design for Protein A [Table 4] Table 5: Turbidity data for the Protein A chromatography process [Table 5]

[0101] In conclusion, the salt concentration and pH of the elution buffer during protein A chromatography elution significantly affect the turbidity of the neutralized sample or protein mixture.

[0102] As shown in Example 1, when protein was eluted with 25 mM acetate buffer, pH 3.5, it showed a significantly higher turbidity of 227 NTU compared to proteins eluted with 125 mM and 200 mM acetate buffer, pH 3.5. Neutralized protein A eluates showed turbidity values ​​of 227.0 NTU and 103.0 NTU, respectively. Comparing 200 mM acetate, pH 3.5 ± 0.1 with 200 mM acetate, pH 3.5 ± 0.1 buffer, neutralized protein A eluates showed turbidity values ​​of 36.1 NTU and 42.5 NTU, respectively. Lower NTU values ​​not only reduce the area required for unit operation but also directly improve subsequent deep filtration performance.

Claims

1. A process for purifying a protein mixture, wherein the process is: a. Loading a protein mixture of antibody or its fragments and impurities onto a protein A chromatography column; b. Washing the protein A chromatography column; c. Optionally, wash with buffer one or more times; d. Elute the protein mixture containing the target antibody with an elution buffer having a pH of 3.0 to 3.5 and a salt concentration of 100 mM to 200 mM; e. Performing a virus inactivation and neutralization step to obtain a neutralized protein mixture, The process consisting of these steps is carried out in this order. The neutralized protein mixture obtained in step (e) above is filtered using a 0.2 μm filter. The neutralized protein mixture has a turbidity value (NTU) measured with a standard turbidimeter that is lower than the turbidity value of the corresponding protein mixture eluted with a buffer with a salt concentration of less than 100 mM. The antibody in question is omalizumab. The process is characterized in that the elution buffer is an acetate buffer.

2. A process according to claim 1, wherein the process is a. Loading a protein mixture of antibody or its fragments and impurities onto a protein A chromatography column; b. Washing the protein A chromatography column; c. Optionally, wash with buffer one or more times; d. Elute the protein mixture containing the target antibody with an elution buffer having a pH of 3.5 ± 0.1 and a salt concentration of more than 125 mM; e. Performing a virus inactivation and neutralization step to obtain a neutralized protein mixture, A process characterized in that the steps comprising the above are carried out in this order, and the neutralized protein mixture has a turbidity of 103 NTU or less, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM, when measured with a standard turbidimeter.

3. A process according to claim 1, wherein the process is a. Loading a protein mixture of antibody or its fragments and impurities onto a protein A chromatography column; b. Washing the protein A chromatography column; c. Optionally, wash with buffer one or more times; d. Elute the protein mixture containing the target antibody with an elution buffer having a pH of 3.5 ± 0.1 and a salt concentration of 200 mM; e. Performing a virus inactivation and neutralization step to obtain a neutralized protein mixture, A process characterized in that the steps comprising the above are carried out in this order, and the neutralized protein mixture has a turbidity of 36.1 NTU or less, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM, when measured with a standard turbidimeter.

4. A process according to claim 1, wherein the process is a. Loading a protein mixture of antibody or its fragments and impurities onto a protein A chromatography column; b. Washing the protein A chromatography column; c. Optionally, wash with buffer one or more times; d. Elute the protein mixture containing the target antibody with a buffer having a pH of 3.0 ± 0.1 and a salt concentration of 200 mM; e. Performing a virus inactivation and neutralization step to obtain a neutralized protein mixture, A process characterized in that the steps comprising the above are carried out in this order, and the neutralized protein mixture has a turbidity of 42.5 NTU or less, which is lower than that of the protein mixture eluted with a buffer having a concentration of less than 100 mM, when measured with a standard turbidimeter.

5. The process according to claim 1, wherein the antibody has an isoelectric point of 6 to 9.

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