Process for the purification of proteins

Abstract

To provide a process for purifying an antibody or a fusion protein from a protein mixture containing product - and process-related impurities.SOLUTION: Provided is a process for purifying a protein of interest from a protein mixture comprising the protein of interest and one or more low molecular weight (LMW) variants, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column with a suitable buffer; b. washing the CHT column with a suitable buffer; and c. eluting the purified protein of interest with a suitable gradient of phosphate buffer, wherein the protein of interest is substantially pure and has less than 0.4% LMW as analyzed by SE-HPLC analysis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to hydroxyapatite chromatography for the purification of at least one antibody or fusion protein from a protein mixture containing low molecular weight impurities and basic variants.

[0002] Furthermore, the present invention provides a scalable purification process for antibodies or fusion proteins by using chromatographic steps. More particularly, the present invention provides purified antibody compositions that are pharmaceutically acceptable and substantially free of product- and process-related impurities. [Background technology]

[0003] Monoclonal antibodies, as a class of therapeutic molecules, are increasingly in demand in the biotechnology industry for the treatment of diseases. Furthermore, these antibodies have heterogeneous biochemical and biophysical properties due to multiple post-translational modifications and degradation events that occur during production. With advances in upstream technologies, monoclonal antibody (mAb) production capacities have increased from a few milligrams per liter to several grams per liter. These titers place significant pressure on downstream processes (DSPs), which must be tailored to achieve higher efficiency and better utilization of available resources. If any of these critical parameters are not determined at the facility design stage, the process can break down, resulting in further commercial losses and delaying the product's entry into the market.

[0004] A key challenge associated with the successful commercialization of antibodies and fusion proteins is developing a pure product with acceptable levels of product- and process-related impurities removed to comply with regulatory requirements. Product- and process-related impurities must be within acceptable limits for approval as set by regulatory agencies. Impurities often include undesirable components such as size and charge variants. The formation of size variants, charge variants, and other undesirable species, such as low molecular weight aggregates (LMW) and basic variants, can adversely affect product safety by causing complement activation or anaphylaxis upon administration. Furthermore, the formation of aggregates and charge variants can hinder the manufacturing process by causing reduced product yield, peak broadening, and loss of activity.

[0005] Hydroxyapatite chromatography has been shown to be a method for the chromatographic purification of monoclonal antibodies (Mabs). Ceramic hydroxyapatite (CHT) chromatography has been reported for the separation of HMW antibodies by using sodium chloride or calcium chloride.

[0006] In the present invention, we have successfully attempted to separate low molecular weight aggregates (LMW) and basic variants (BV) from antibody preparations using ceramic hydroxyapatite chromatography.

[0007] In this invention, we also focus on other chromatographic techniques to separate other process and product related impurities such as high molecular weight aggregates (HMW) and acidic variants.

[0008] For example, it has been reported that ion exchange and hydrophobic interaction chromatography can induce aggregate formation due to increased protein concentration or necessary changes in buffer concentration and / or pH during elution. Furthermore, in some cases, antibodies exhibit isoelectric point differences that are too small to allow separation by ion exchange chromatography (Non-Patent Document 1). However, the present invention uses anion exchange, which significantly reduces high molecular weight impurities and acidic variants.

[0009] Size exclusion chromatography is tedious and leads to highly diluted products, which is an obstacle to large-scale efficiency-based manufacturing processes. Ligand leakage from affinity chromatography columns can also occur, resulting in undesirable contamination of the eluted product (Non-Patent Document 2).

[0010] There is currently a need for methods to produce and purify antibodies of interest in a sufficiently pure form suitable for pharmaceutical use. The present invention addresses this need.

[0011] The present invention provides a scalable and robust purification process that significantly reduces antibody or fusion protein-associated impurities. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Tarditi, J. Immunol. Methods 599:13-20 (1992) [Non-patent document 2] Steindl, J. Immunol. Methods 235:61-69 (2000) Summary of the Invention [Problem to be solved by the invention]

[0013] In one embodiment, the present invention provides a simple and scalable CHT process that removes at least one impurity selected from LMW and basic variants by using a phosphate elution gradient without the use of NaCl, CaCl, or other additives.

[0014] In one aspect of such embodiments, the purified protein mixture is free of, or contains minimally acceptable amounts of, process and product related impurities such as host cell proteins, host cell DNA, leached proteins, half antibodies, clipped antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weight (HMW) species.

[0015] In one embodiment, the purification process provides a purity of the monomer selected from greater than 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% purity.

[0016] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more low molecular weight (LMW) variants, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis.

[0017] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more basic variants (BVs), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

[0018] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest, one or more low molecular weight (LMW) variants, and one or more basic variants (BV), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, the purified protein of interest is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis; The process provides that the purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

[0019] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0020] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0021] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant 2H1L, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has less than 3% 2H1L as measured by CE-SDS.

[0022] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises low 2H1L selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, or 1.4% or less.

[0023] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant HH, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has less than 0.2% HH as measured by CE-SDS.

[0024] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low HH selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less, or 0.08% or less, 0.06% or less, or 0.04% or less, and 0.03% or less.

[0025] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant HC, the process comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an HC of less than 0.4 as measured by CE-SDS.

[0026] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low HC selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less, and 0.18% or less.

[0027] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant LC, the process comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an LC reduced to 0.5% as measured by CE-SDS.

[0028] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low LC selected from about 0.5% or less, 0.4% or less, 0.3% or less, and 0.2 or less.

[0029] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and low molecular weight (LMW) variants LC, HC, HH, and 2H1L, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides a purified protein of interest that is substantially pure and has up to 3% LMW, HC, HH, and 2H1L reduced to 3% as measured by CE-SDS. In one aspect of such an embodiment, the protein mixture eluted from the CHT column contains 2.2% to 2.6% LMW selected from LC, HC, HH, and 2H1L as measured by CE-SDS.

[0030] In another embodiment, the process provides a linear elution gradient on the CHT column in which the phosphate concentration is gradually increased from about 32 mM to 88 mM to separate or reduce LMW having a molecular weight of about 23 kDa to about 125 kDa and reduce basic variants by about 33%.

[0031] In another embodiment, the process provides a linear elution gradient on a CHT column in which the phosphate concentration is gradually increased from about 40 mM to 96 mM to separate or reduce LMW having a molecular weight of about 23 kDa to about 125 kDa and reduce basic variants by about 33%.

[0032] In another embodiment, the present invention provides a 50 L, 100 L, 200 L scalable purification process that can provide a substantially pure monomeric form of an antibody or fusion protein and a low tolerance of impurities selected from host cell proteins, host cell DNA, leached proteins, half antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weight (HMW) species.

[0033] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and acidic species or variants thereof, comprising: a. purifying the protein mixture by affinity chromatography Protein A or Protein G; b. subjecting the protein mixture obtained by affinity chromatography to viral inactivation; c. loading the protein mixture obtained in step (b) onto an anion exchange resin with a suitable buffer at a suitable pH selected from pH 7.0 to 7.5; d. eluting the protein mixture in a flow-through mode to allow acidic species or variants of the protein of interest to bind to the anion exchange resin; Including, wherein the eluted protein mixture obtained in step (d) comprises a substantially purified protein of interest and less than 15% acidic species or variants when analyzed by CEX-HPLC analysis.

[0034] In one aspect of such an embodiment, the process provides the protein mixture comprising the acidic variant at about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, or less than 1% AV.

[0035] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and high molecular weight (HMW) impurities, the process comprising: a. purifying the protein mixture by affinity chromatography Protein A or Protein G; b. subjecting the protein mixture obtained by affinity chromatography to viral inactivation; c. loading the protein mixture obtained in step (b) onto an anion exchange resin with a suitable buffer at a suitable pH selected from pH 7.0 to 7.5; d. eluting the protein mixture in a flow-through mode to bind HMW impurities to the anion exchange resin; Including, wherein the eluted protein mixture obtained in step (d) comprises a substantially purified protein of interest and less than 0.5% HMW impurities when analyzed by SE-HPLC analysis.

[0036] In one aspect of such an embodiment, the process provides a protein mixture containing less than 0.5%, about 0.4% or less, or 0.3% or less, or 0.2% or less, or 0.1% or less HMW.

[0037] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture, comprising: a. obtaining a protein mixture from a mammalian expression system containing the protein of interest and at least one impurity selected from an acidic variant, a basic variant, a low molecular weight (LMW), and a high molecular weight (HMW); b. applying the protein mixture to an affinity chromatography column; c. eluting the protein mixture from the affinity chromatography column; d. performing viral inactivation of the protein mixture obtained in step (c); e. applying the protein mixture obtained in step (d) to anion exchange chromatography; f. eluting the protein mixture in a flow-through mode; g. applying the protein mixture obtained in step (f) to a ceramic hydroxyapatite (CHT) column; h. optionally washing the CHT column with an appropriate wash buffer; i. eluting the protein mixture from the CHT column with a suitable buffer. In certain embodiments, the protein of interest is an IgG1 antibody or a fusion protein, wherein the eluted protein mixture is enriched in the protein of interest and is substantially free of impurities such as HMW, LMW, acidic, and basic variants (BV).

[0038] In one embodiment, the protein of interest is an IgG1 antibody that binds to IgE. In a preferred embodiment, the IgG1 antibody is a biosimilar of omalizumab. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 shows the process chromatogram of the AEX run. [Figure 2] Figure 2 shows the CEX-HPLC chromatogram (charge variants) of the AEX run. [Figure 3]Figure 3 shows the SE-HPLC chromatograms (size variants) of the AEX run. [Figure 4] Figure 4 shows a magnified view of the SE-HPLC chromatogram (size variants) of the AEX run. [Figure 5] Figure 5 shows the process chromatogram of the CHT run. [Figure 6] Figure 6 shows the SE-HPLC chromatograms (size variants) of the CHT run. [Figure 7] Figure 7 shows the CEX-HPLC chromatogram (charge variants) of the CHT run.

[0040] The present invention provides a purification process that can reduce various product and process related impurities.

[0041] In certain embodiments, the present invention provides for the reduction of a few product-related impurities through the use of CHT chromatography.

[0042] In certain embodiments, the present invention provides for the reduction of a few product-related impurities through the use of anion exchange chromatography.

[0043] In certain embodiments, the present invention provides for the reduction of product-related impurities through the use of CHT and anion exchange chromatography.

[0044] In certain embodiments, the present invention provides for the reduction of product-related impurities through the use of affinity chromatography, CHT, and anion exchange chromatography.

[0045] The term "antibody" includes immunoglobulin molecules composed of four polypeptide chains, two heavy (H) and two light (L) chains, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0046] Omalizumab (Xolair®) is a recombinant DNA-derived humanized IgG1K monoclonal antibody that selectively binds to human immunoglobulin E (IgE). The antibody has a molecular weight of approximately 149 kD. Xolair® is manufactured by suspension culture of Chinese hamster ovary cells in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product. Xolair® is a sterile, white, preservative-free lyophilized powder contained in single-use vials and is administered as a subcutaneous (SC) injection after reconstitution with Sterile Water for Injection (SWFI) USP.

[0047] As used herein, the terms "acidic variant" or "acidic species" and "AV" refer to a variant of a protein, e.g., an antibody or antigen-binding portion thereof, characterized by an overall acidic charge. For example, in monoclonal antibody (mAb) preparations, such acidic species can be detected by various methods, such as ion exchange, e.g., WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Acidic variants of antibodies are formed by chemical and enzymatic modifications, such as deamidation and sialylation, respectively, which result in an increase in the net negative charge and a decrease in the pI value of the antibody, thereby forming the acidic variant. Degradation of C-terminal lysines results in the loss of the net positive charge, leading to the formation of the acidic variant. Another mechanism for generating acidic variants is the formation of various covalent adducts, such as glycation, in which glucose or lactose can react with the primary amines of lysine residues during production in glucose-rich media or during storage if reducing sugars are present in the formulation (MAbs. 2010 Nov-Dec; 2(6):613-624).

[0048] The term "acidic variant" does not include process-related impurities. As used herein, the term "process-related impurities" refers to impurities that are present in a composition containing a protein but are not derived from the protein itself. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatography materials, and media components.

[0049] As used herein, the term "anion exchange chromatography," or "anion exchange column," or "AEX," refers to a form of ion exchange chromatography (IEX) used to separate molecules based on net surface charge. Anion exchange chromatography, more specifically, uses a positively charged ion exchange resin that has an affinity for molecules with a net negative surface charge. Anion exchange chromatography is used for both preparative and analytical purposes and can separate a wide range of molecules, from amino acids and nucleotides to large proteins. Here, we focus on preparative anion exchange chromatography of proteins.

[0050] As used herein, the term "POROS 50 HQ" refers to a Thermo Scientific TM POROS TM A strong anion exchange resin (POROS AEX resin) designed for charge-based chromatographic separation of biomolecules, including recombinant proteins and monoclonal antibodies. Thermo Scientific TM POROS TM The 50HQ resin is functionalized with quaternized polyethyleneimine groups.

[0051] When "strong anion exchange" is used in a flow-through process, the equilibrium is shifted, and impurities are differentiated from the protein of interest. Strong anion exchange is commonly known for removing Protein A contaminants, HCPs, DNA, or viruses in antibody purification. In a flow-through protocol, the sample and equilibration buffer are adjusted to conditions where contaminating molecules still bind to the resin, but the protein of interest (due to charge) does not. This is accomplished by increasing the salt concentration and / or raising the buffer pH to a point below the pI of the molecule of interest.

[0052] As used herein, the term "flow-through mode" or "flow-through" refers to a purification process in which the antibody of interest does not bind to the chromatography resin. In certain embodiments, at least 50% of the antibody of interest does not bind to the chromatography resin. In certain embodiments, at least 60%, 70%, or 80% of the antibody of interest does not bind to the chromatography resin. However, process- and product-related impurities do bind to the chromatography resin. In certain embodiments, at least 50% of the process- and product-related impurities do bind to the chromatography resin. In certain embodiments, at least 60%, 70%, or 80% of the process- and product-related impurities do bind to the chromatography resin.

[0053] As used herein, the terms "column" or "resin" or "chromatography resin or chromatography column" are interchangeable.

[0054] The term "CHT" or "ceramic hydroxyapatite chromatography" refers to a form of calcium phosphate used in the chromatographic separation of biomolecules. Sets of five calcium doublets (C sites) and pairs of -OH-containing phosphate triplets (P sites) are arranged in a repeating geometric pattern. Electron micrographs of this material show a repeating hexagonal structure. Space-filling models and repeating structures have also been constructed using Raman spectroscopy. Hydroxyapatite has unique separation properties and unparalleled selectivity and resolution. It often separates proteins that have been shown to be homogeneous by electrophoresis and other chromatographic techniques.

[0055] Applications of hydroxyapatite chromatography include the purification of various subclasses of monoclonal and polyclonal antibodies, antibodies with different light chain compositions, antibody fragments, isozymes, supercoiled DNA from linear duplexes, and single-stranded DNA from double-stranded DNA. CHT ceramic hydroxyapatite is a spherical macroporous form of hydroxyapatite. Upon sintering at high temperatures, it transforms from a crystalline to a ceramic form.

[0056] As used herein, the term "substantially pure antibody" includes an antibody that is substantially free of impurities selected from product- or process-related impurities. In certain embodiments, the antibody is free of acidic variants, basic variants, low molecular weight, and high molecular weight, and a substantially pure antibody has a purity of less than about 99%, or less than about 98%, or less than about 97%, or less than about 95%, or less than about 92%, or less than about 90%, or less than about 88%, or less than about 85%, or less than about 82%, or less than about 80%, or less than about 75%, or less than about 70%, or less than about 65%, or less than about 60%, or less than 50%.

[0057] The term used as "size variant" refers to LMW, HMW, or aggregates.

[0058] The term "low molecular weight" or "LMW" species refers to fragments of the protein backbone that are cleaved and are considered product-related impurities that contribute to the size heterogeneity of antibodies. LMW species often have low or significantly reduced activity relative to monomeric antibodies and may contribute to immunogenicity or affect pharmacokinetic properties in vivo. As a result, LMW species are considered critical quality attributes that are routinely monitored during drug development as part of release testing of purified drug products during manufacturing. The LMW has a molecular weight selected from 23 kDa, 24 kDa, 25 kDa, 26 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 126 kDa, and 127 kDa. In certain embodiments, the LMW is selected from LC, HC, HH, and 2HL1.

[0059] The term "light chain" or "LC" of an antibody, which has a weight of 25 kDa, refers to a light or L chain. A typical antibody contains two light chains: lambda (λ) and kappa (κ). Antibodies have either κ or λ chains, but not one of each. There is no functional difference between antibodies with λ or κ light chains, and both types of light chains are found in antibodies of all five major classes. The ratio of the two types of light chains varies by species. However, protein mixtures may contain only one LC, which is considered an LMW impurity.

[0060] The term "heavy chain" or "HC" of an antibody refers to a heavy or heavy chain, each weighing 50 kDa. There are several different types of heavy chains that define the class or isotype of an antibody. These heavy chain types vary depending on the animal. The heavy chain contains a series of immunoglobulin domains, including one variable domain (VH) for binding to an antigen and constant domains (CH1, CH2, etc.). α and γ heavy chains have approximately 450 amino acids, while μ and ε heavy chains have approximately 550 amino acids. However, a protein mixture may contain only one HC, which is considered an LMW impurity.

[0061] The term "Two Heavy One Light Chain," or "2H1L," or "H2L," refers to an antibody species having a single light chain. 2H1L species of antibodies are formed by beta-elimination of heavy chain cysteine ​​residues that form the heavy chain-light chain disulfide bond, resulting in the formation of the H2L species.

[0062] The term "high molecular weight" or "HMW" refers to a product-related impurity that results in size heterogeneity in antibody products. The generation of HMW species in therapeutic antibody pharmaceuticals as a result of protein aggregation can compromise both the efficacy and safety of the drug (e.g., by eliciting an undesirable immunogenic response). HMW is considered a critical quality attribute that is routinely monitored during drug development as part of release testing of purified pharmaceuticals during manufacturing. In certain embodiments, HMW refers to aggregates.

[0063] The term "aggregate" is classified based on the type of interaction and solubility. Soluble aggregates are invisible particles that cannot be removed by filtration. Insoluble aggregates can be removed by filtration and are often visible to the human eye. Both types of aggregates pose problems in biopharmaceutical development. Covalent aggregates arise from the formation of covalent bonds between multiple monomers of a given peptide. Disulfide bond formation of free thiols is a common mechanism of covalent aggregation. Oxidation of tyrosine residues can lead to the formation of bityrosines, which often result in aggregation. Reversible protein aggregation is usually due to weaker protein interactions, such as dimers, trimers, and multimers.

[0064] The term "basic variant" refers to variants resulting from the presence of a C-terminal lysine or amidation of glycine, formation of succinimide, oxidation of amino acids, or removal of sialic acid, which introduce an additional positive charge or remove a negative charge; any type of modification increases the pI value.

[0065] The term "high salt buffer" as used herein refers to a high strength or high molarity buffer.

[0066] The terms used as "Buffer A" and "Buffer B" are interchangeable with "first buffer" or "second buffer," respectively, in CHT chromatography.

[0067] As used herein, the expression "virus reduction / inactivation" 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, but not limited to, a reduction in their infectivity or replicative ability ("inactivation"). Such a 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%.

[0068] As used herein, "comprises" or "comprising" does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is to be considered as any embodiment of the term "comprising of". Hereinafter, if a group is defined to include at least a certain number of embodiments, this is understood to also disclose a group that may consist only of these embodiments.

[0069] Throughout the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0070] As used herein, the term "about" is intended to mean a range of about 10-20% greater or less than a reference value. In certain circumstances, depending on the nature of the reference value, one of ordinary skill in the art will recognize that the term "about" can mean a value greater or less than a 10-20% deviation from that value.

[0071] As used herein, the term "CE-SDS" is used to analyze LMW selected from LC, HC, HH, and 2HL1.

[0072] The term "analytical HPLC" includes CEX HPLC, which is used for estimation of charge variants, and SE-HPLC, which is used for estimation of HMW and LMW.

[0073] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more low molecular weight (LMW) variants, comprising: a. loading the protein mixture in an appropriate buffer onto a ceramic hydroxyapatite (CHT) column; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with a suitable gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis.

[0074] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0075] In one embodiment, the LMW has a molecular weight selected from 23 kDa, 24 kDa, 25 kDa, 26 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115 kDa, 120 kDa, 125 kDa, 126 kDa, and 127 kDa.

[0076] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more basic variants (BVs), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

[0077] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0078] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest, one or more low molecular weight (LMW) variants, and one or more basic variants (BV), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, the purified protein of interest is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis; The process provides that the purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

[0079] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0080] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0081] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant 2H1L, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has less than 3% 2H1L as measured by CE-SDS.

[0082] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises low 2H1L selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, or 1.4% or less.

[0083] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant HH, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has less than 0.2% HH as measured by CE-SDS.

[0084] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low HH selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less, or 0.08% or less, 0.06% or less, or 0.04% or less, and 0.03% or less.

[0085] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant HC, the process comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an HC of less than 0.4 as measured by CE-SDS.

[0086] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low HC selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less, and 0.18% or less.

[0087] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and a low molecular weight (LMW) variant LC, the process comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an LC reduced to 0.5% as measured by CE-SDS.

[0088] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low LC selected from about 0.5% or less, 0.4% or less, 0.3% or less, and 0.2 or less.

[0089] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and low molecular weight (LMW) variants LC, HC, HH, and 2H1L, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has LC, HC, HH, and 2H1L reduced to 3% as measured by CE-SDS.

[0090] In one aspect of such an embodiment, the protein mixture eluted from the CHT column contains 2.2% to 2.6% of the LMW LC, HC, HH, and 2H1L as measured by CE-SDS.

[0091] In another embodiment, the process provides a linear elution gradient on the CHT column in which the phosphate concentration is gradually increased from about 32 mM to 88 mM to separate or reduce LMW having a molecular weight of about 23 kDa to about 125 kDa and reduce basic variants by about 33%.

[0092] In another embodiment, the process provides a linear elution gradient on a CHT column in which the phosphate concentration is gradually increased from about 40 mM to 96 mM to separate or reduce LMW having a molecular weight of about 23 kDa to about 125 kDa and reduce basic variants by about 33%.

[0093] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest, one or more low molecular weight (LMW) variants, and one or more basic variants (BV), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis.

[0094] In one embodiment, the purification process reduces the LMW by at least about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, and less than about 50%.

[0095] In one embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more basic variants (BVs), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process provides that the purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

[0096] In one embodiment, the purification process reduces basic variants by at least about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%, about 35%, about 40%, or about 45%, and about 50%.

[0097] In a preferred embodiment, the purification process reduces basic variants by at least about 33%.

[0098] In another embodiment, the purification process reduces basic variants by at least about 33% at a 5 L, or 50 L, or 100 L, or 200 L scale.

[0099] In another embodiment, the present invention provides a purification process scalable at 5 L, 50 L, 100 L, and 200 L that can provide a substantially pure monomeric form of an antibody or fusion protein and a low tolerance of impurities selected from host cell proteins, host cell DNA, leached proteins, half antibodies, dimers, tetramers, acidic or basic charge variants, aggregates, low molecular weight (LMW) species, and high molecular weight (HMW) species.

[0100] The present invention also provides reliable product quality by providing a robust, standardized purification process that can be used with the same efficiency and provide uniform results from small-scale (5 L) to large-scale (50 L to 200 L or more) purification. An additional advantage is the development of effective, reliable, and regulatory (e.g., in terms of U.S. FDA and / or EMA) compliant proteins of interest, such as antibodies or fusion proteins.

[0101] In certain embodiments, the steps of loading, washing, and eluting the CHT column are completed within about 30 minutes to about 105 minutes, and in certain embodiments, the steps of loading, washing, and eluting the CHT column are completed within 104 minutes.

[0102] In a particular embodiment, the mammalian cell is a CHO cell.

[0103] In one embodiment, the load, wash, and elution buffers do not contain an additive selected from sodium chloride, calcium chloride, and glycine.

[0104] In one embodiment, the suitable buffer is a polar protic molecule, sodium phosphate, Tris, HEPES, or the like.

[0105] In one embodiment, the load, wash, and elution buffers have a pH in the range of about 6.5 to about 7.2.

[0106] In one embodiment, the load, wash, and elution buffers have a pH of about 7.0±0.2.

[0107] In one embodiment, the load and wash buffers have a conductivity in the range of about 3 to about 7 mS / cm.

[0108] In a preferred embodiment, the load and wash buffers have a conductivity of about 6 mS / cm.

[0109] In another embodiment, the elution phosphate gradient is a linear gradient in which the concentration of phosphate gradually increases from about 5% to 25% buffer B.

[0110] In another embodiment, the phosphate concentration is gradually increased from about 8% to 22%.

[0111] In another embodiment, the phosphate concentration is gradually increased from about 10% to 24%.

[0112] In one aspect of such an embodiment, the protein mixture eluted from the CHT column contains less than 0.4% LMW when analyzed by SE-HPLC analysis.

[0113] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0114] In one aspect of such an embodiment, the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0115] In one aspect of such an embodiment, the protein mixture eluted from the CHT column contains less than 10% BV.

[0116] In another embodiment, the process provides a linear elution gradient in which the concentration of phosphate gradually increases from about 24 mM to 100 mM.

[0117] In another embodiment, the process provides a linear elution gradient on a CHT column in which the phosphate concentration gradually increases from about 32 mM to 88 mM.

[0118] In another embodiment, the process provides a linear elution gradient on a CHT column in which the phosphate concentration gradually increases from about 40 mM to 96 mM.

[0119] In one embodiment, the present invention provides a process for purifying an antibody that binds IgE from a protein mixture comprising said antibody, one or more low molecular weight (LMW) variants, and one or more basic variants (BV), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. Eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, the purified antibody is substantially pure and has an LMW of less than 0.4% when analyzed by SE-HPLC analysis; The process provides that the purified antibody is substantially pure and contains less than 15% basic variants when analyzed by CEX-HPLC analysis.

[0120] In one aspect of such an embodiment, the antibody eluted from the CHT column contains an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0121] In one aspect of such an embodiment, the antibody eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0122] In certain embodiments, the protein of interest is an IgG1 antibody or a fusion protein. In one embodiment, the protein of interest is an IgG1 antibody that binds to IgE. In a preferred embodiment, the IgG1 antibody is a biosimilar of omalizumab.

[0123] In one embodiment, the purification process provides a monomer purity selected from greater than 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%.

[0124] In one embodiment, the present invention provides a purified pharmaceutical composition of omalizumab comprising said substantially purified monomer of omalizumab and less than about 0.2% LMW as measured by SE-HPLC.

[0125] In another embodiment, the present invention provides a purified pharmaceutical composition of omalizumab purified by CHT chromatography, comprising a substantially purified monomer of said omalizumab and less than about 0.2% LMW as measured by SE-HPLC.

[0126] In another embodiment, the present invention provides a purified pharmaceutical composition of omalizumab purified by CHT chromatography, comprising the substantially purified monomer of omalizumab and 2.2% to 2.6% of an LMW selected from LC, HC, HH, and 2H1L, as measured by CE-SDS.

[0127] In another embodiment, the present invention provides a purified pharmaceutical composition of Omalizumab comprising a substantially purified monomer of said Omalizumab and less than 5% of a basic variant as measured by CEX-HPLC.

[0128] In another embodiment, the present invention provides a purified pharmaceutical composition of Omalizumab purified by CHT chromatography, comprising a substantially purified monomer of said Omalizumab and less than 5% of a basic variant as measured by CEX-HPLC.

[0129] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and an acidic species or variant thereof, comprising the steps of: a. loading the protein mixture onto an anion exchange resin in a suitable buffer at a suitable pH selected from pH 7.0 to 7.5; b. eluting the protein mixture in a flow-through mode to allow acidic species or variants of the protein of interest to bind to the anion exchange resin; Including, The process provides a method for preparing a soluble protein mixture, the method comprising: eluting the eluted protein mixture and eluting the eluted protein; eluting the eluted protein mixture; and eluting the eluted protein ....

[0130] In one aspect of such an embodiment, the process provides the protein mixture comprising the acidic variants at about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, or less than 1% AV.

[0131] In another embodiment, the present invention provides a purified pharmaceutical composition of Omalizumab comprising a substantially purified monomer of said Omalizumab and less than 12% of an acidic variant as measured by CEX-HPLC.

[0132] In another embodiment, the present invention provides a purified pharmaceutical composition of omalizumab purified by AEX chromatography, comprising a substantially purified monomer of said omalizumab and less than 12% acidic variants as measured by CEX-HPLC.

[0133] In another embodiment, the present invention provides a purified pharmaceutical composition of omalizumab purified by AEX chromatography, comprising a substantially purified monomer of said omalizumab and less than 10% acidic variants as measured by CEX-HPLC.

[0134] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and high molecular weight (HMW) impurities, the process comprising: a. loading the obtained protein mixture onto an anion exchange resin with an appropriate buffer at an appropriate pH selected from pH 7.0 to 7.5; b. eluting the protein mixture in a flow-through mode to bind the HMW impurities to the anion exchange resin; Including, The process provides that the eluted protein mixture contains substantially pure monomeric form of the protein of interest and less than 0.5% of the HMW impurities when analyzed by SE-HPLC analysis.

[0135] In one aspect of such an embodiment, the process provides a protein mixture containing less than 0.5%, about 0.4% or less, or 0.3% or less, or 0.2% or less, or 0.1% or less HMW.

[0136] In another embodiment, the present invention provides a purified pharmaceutical composition of omalizumab purified by AEX chromatography, comprising a substantially purified monomer of said omalizumab and less than 0.5% HMW as measured by SE-HPLC.

[0137] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and an acidic species or variant thereof, comprising the steps of: a. purifying the protein mixture by affinity chromatography Protein A or Protein G; b. subjecting the protein mixture obtained by affinity chromatography to viral inactivation; c. loading the protein mixture obtained in step (b) onto an anion exchange resin with a suitable buffer at a suitable pH selected from pH 7.0 to 7.5; d. eluting the protein mixture in a flow-through mode to allow acidic species or variants of the protein of interest to bind to the anion exchange resin; Including, wherein the eluted protein mixture obtained in step (d) comprises substantially pure monomeric form of the protein of interest and less than 15% acidic species or variants when analyzed by CEX-HPLC analysis.

[0138] In one aspect of such an embodiment, the process provides the protein mixture comprising the acidic variants at about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, or less than 1% AV.

[0139] In one aspect of such embodiment, the purification process reduces acidic variants by at least 40%, preferably 50%, in a protein mixture obtained in flow-through mode on strong anion exchange.

[0140] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture comprising said protein of interest and high molecular weight (HMW) impurities, the process comprising: a. purifying the protein mixture by affinity chromatography Protein A or Protein G; b. subjecting the protein mixture obtained by affinity chromatography to viral inactivation; c. loading the protein mixture obtained in step (b) onto an anion exchange resin with a suitable buffer at a suitable pH selected from pH 7.0 to 7.5; d. eluting the protein mixture in a flow-through mode to bind the HMW impurities to the anion exchange resin; Including, wherein the eluted protein mixture obtained in step (d) comprises substantially pure monomeric form of the protein of interest and less than 0.5% HMW impurities when analyzed by SE-HPLC analysis.

[0141] In one aspect of such an embodiment, the process provides a protein mixture containing less than 0.5%, about 0.4% or less, or 0.3% or less, or 0.2% or less, or 0.1% or less HMW.

[0142] In one aspect of such an embodiment, the purification process reduces HMW by at least 80%, preferably 90%, in a protein mixture obtained in flow-through mode on strong anion exchange.

[0143] In one embodiment, the present invention surprisingly found that acidic variants and HMWs are removed through a strong anion exchange column by running the column in flow-through mode, where the pH of the buffer solution is 7.0-7.3, slightly lower than the pI of omalizumab. By optimizing the desired pH of the buffer, at least 40% or more of the acidic variants or HMWs are substantially bound to the strong anion exchange. In certain embodiments, more than 80% of the HMWs are bound to the strong anion exchange.

[0144] In one embodiment, the invention provides a purified antibody composition obtained from strong anion exchange having less than 15%, preferably less than 12%, acidic variants and less than 0.5%, preferably 0.3%, HMW, which is below the limits accepted by regulatory agencies.

[0145] In one embodiment, the present invention is highly useful in reducing downstream processing burdens by eliminating the use of multiple columns, and in certain embodiments, the present invention does not use HIC and multimodal chromatography.

[0146] In another embodiment, the present invention provides a process for purifying a protein of interest from a protein mixture, comprising: a. obtaining a protein mixture from a mammalian expression system containing the protein of interest and at least one impurity selected from an acidic variant, a basic variant, a low molecular weight (LMW), and a high molecular weight (HMW); and applying the protein mixture to an affinity chromatography column; b. eluting the protein mixture from the affinity chromatography column; c. performing viral inactivation of the protein mixture obtained in step (c); d. applying the protein mixture obtained in step (d) to anion exchange chromatography; e. eluting the protein mixture in a flow-through mode; f. applying the protein mixture obtained in step (f), which is substantially free of HMW and acidic variants, to a ceramic hydroxyapatite (CHT) column; g. Optionally, washing the CHT column with an appropriate wash buffer; h. Eluting the protein mixture from the CHT column with an appropriate buffer; Including, The process provides that the eluted protein mixture is enriched in the protein of interest and is substantially free of HMW, LMW, and basic variant (BV) impurities.

[0147] In one embodiment, the invention provides an anion exchange process that produces an antibody composition enriched for the antibody of interest and substantially reduced in acidic variants and HMW or aggregates.

[0148] In one embodiment, the invention provides an anion exchange process conducted in a negative mode to produce an antibody composition that is enriched for the antibody of interest and substantially reduced in acidic variants, comprising about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, or 1% or less AV.

[0149] In one embodiment, the invention provides an anion exchange process conducted in a negative mode that enriches the antibody of interest and substantially reduces HMW content, producing an antibody composition comprising about 0.5% or less HMW. In one aspect of this embodiment, the low HMW composition comprises about 0.4% or less HMW, 0.3% or less HMW, 0.2% or less HMW, or 0.1% or less HMW.

[0150] In one embodiment, the present invention provides a CHT column chromatography process that produces an antibody composition enriched for the antibody of interest and reduced in basic variants and LMW impurities.

[0151] In one embodiment, the present invention provides a protein mixture eluted from a CHT column comprising an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0152] In one embodiment, the present invention provides a protein mixture eluted from a CHT column containing less than 0.4% LMW when analyzed by SE-HPLC analysis.

[0153] In one embodiment, the present invention provides a protein mixture eluted from a CHT column comprising a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0154] In one embodiment, the present invention provides a protein mixture eluted from a CHT column that contains less than 10% BV.

[0155] In one embodiment, the present invention provides a protein mixture eluted from a CHT column that contains less than 5% BV.

[0156] In another embodiment, the present invention provides a process for purifying omalizumab from a protein mixture at a 50 L scale or a 200 L scale, comprising: a. obtaining a protein mixture from a mammalian expression system containing a protein of interest and at least one impurity selected from an acidic variant, a basic variant, a low molecular weight (LMW), and a high molecular weight (HMW); b. applying the protein mixture to an affinity chromatography column; c. eluting the protein mixture from the affinity chromatography column; d. performing viral inactivation of the protein mixture obtained in step (c); e. applying the protein mixture obtained in step (d) to anion exchange chromatography; f. eluting the protein mixture in a flow-through mode; g. applying the protein mixture obtained in step (f), which is substantially free of HMW and acidic variants, to a ceramic hydroxyapatite (CHT) column; h. washing the CHT column with an appropriate wash buffer; i. eluting the protein mixture from the CHT column with an appropriate buffer; Including, The process provides that the eluted protein mixture is enriched with omalizumab and is substantially free of the impurities LMW and basic variants (BV).

[0157] In one embodiment, the CHT wash removes loosely bound impurities.

[0158] In one embodiment, the invention provides an anion exchange process that produces an omalizumab composition enriched in the antibody of interest and substantially reduced in acidic variants and HMW or aggregates.

[0159] In one embodiment, the invention provides an anion exchange process conducted in negative mode to produce an antibody composition enriched in omalizumab and substantially reduced in acidic variants, comprising about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, or 1% or less AV.

[0160] In one embodiment, the invention provides an anion exchange process conducted in negative mode to produce an antibody composition that is enriched in omalizumab and substantially reduced in HMW, comprising about 0.5% or less HMW. In one aspect of this embodiment, the low-HMW composition comprises about 0.4% or less HMW, 0.3% or less HMW, 0.2% or less HMW, or 0.1% or less HMW, as analyzed by SE-HPLC analysis.

[0161] In one embodiment, the present invention provides a CHT column chromatography process for producing an antibody composition enriched for omalizumab and reduced in basic variants and LMW impurities.

[0162] In one embodiment, the present invention provides a protein mixture eluted from a CHT column comprising an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

[0163] In one embodiment, the present invention provides a protein mixture eluted from a CHT column containing less than 0.4% LMW when analyzed by SE-HPLC analysis.

[0164] In one embodiment, the present invention provides a protein mixture eluted from a CHT column comprising a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

[0165] In one embodiment, the present invention provides a protein mixture eluted from a CHT column that contains less than 10% BV.

[0166] In one embodiment, the present invention provides a protein mixture eluted from a CHT column that contains less than 5% BV.

[0167] In one embodiment, the antibody is capable of binding to IgE, thereby treating allergic diseases, asthma, nasal polyps, and hives. In a preferred embodiment, the antibody is omalizumab.

[0168] In one embodiment, a purified pharmaceutical composition of a biosimilar of omalizumab comprising substantially purified monomer and the impurities less than 12% acidic variants, less than 5% basic variants, less than 0.4% LMW, and less than 0.5% HMW.

[0169] In one embodiment, the antibody is selected from IgG1, IgG2, IgG3, IgG4, and fusion proteins. In a specific embodiment, the antibody is selected from etanercept, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, eculizumab, certolizumab pegol, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, trastuzumab emtansine, raxidis, ribozyme, ribozyme, ribozyme, ribozyme 1, ribozyme 2, ribozyme 3, ribozyme 4, ribozyme 5, ribozyme 6, ribozyme 7, ribozyme 8, ribozyme 9, ribozyme 10, ribozyme 11, ribozyme 12, ribozyme 13, ribozyme 14, ribozyme 15, ribozyme 16, ribozyme 17, ribozyme 18, ribozyme 19, ribozyme 20, ribozyme 21, ribozyme 22, ribozyme 23, ribozyme 24, ribozyme 25, ribozyme 26, ribozyme 27, ribozyme 28, ribozyme 29, ribozyme 30, ribozyme 31, ribozyme 32, The drug is selected from bacumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, dalucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, sarilumab, ocrelizumab, tildrakizumab, romosozumab, brolucizumab, and crizanlizumab.

[0170] In one embodiment, the protein of interest is an IgG1 antibody that binds to IgE. In a preferred embodiment, the IgG1 antibody is a biosimilar of omalizumab.

[0171] In one embodiment, the sequential chromatographic steps following affinity chromatography are coordinated or interchangeable with one another. In certain embodiments, affinity chromatography can be followed by CHT chromatography. In certain embodiments, CHT can be performed between affinity chromatography and AEX chromatography.

[0172] In certain embodiments, affinity chromatography can be followed by CHT.

[0173] In certain embodiments, CHT can be performed between affinity chromatography and AEX chromatography.

[0174] Affinity chromatography conditions:

[0175] In one embodiment, the affinity chromatography is selected from Protein A or Protein G.

[0176] In one embodiment, the affinity chromatography resin is selected from Mabselect, Mabselect SuRe, Mabselect SuRe LX, Prosep Ultra Plus, Eshmuno A. In a preferred embodiment, the affinity chromatography resin is Mabselect Sure LX.

[0177] In one embodiment, the equilibration buffer, or loading buffer, or washing buffer is selected from sodium phosphate, Tris-HCl, Tris-acetate, HEPES, glycine-NaOH.

[0178] In a preferred embodiment, the loading buffer is Tris-acetate or Tris-HCl.

[0179] In certain embodiments, the equilibration buffer, loading buffer, or washing buffer is used in combination with a salt. In certain embodiments, the salt is selected from sodium chloride and potassium chloride. In a preferred embodiment, the salt is sodium chloride.

[0180] In one embodiment, the equilibration buffer has a concentration ranging from about 5 mM to about 40 mM. In a specific embodiment, the equilibration buffer has a concentration ranging from about 10 mM to about 25 mM. In a preferred embodiment, the equilibration buffer has a concentration of about 20 mM.

[0181] In certain embodiments, the equilibration buffer, load buffer, or wash buffer optionally comprises a salt selected from about 50 mM to about 400 mM. In one embodiment, the equilibration buffer comprises a salt buffer concentration selected from about 100 mM to about 200 mM. In one embodiment, the equilibration buffer concentration is about 150 mM. In another embodiment, the equilibration buffer concentration is about 100 mM.

[0182] In one embodiment, the equilibration buffer, load buffer, or wash buffer has a conductivity range of about 10 mS / cm to about 20 mS / cm. In one embodiment, the conductivity of the equilibration buffer, load buffer, or wash buffer is about 15.0 mS / cm to 18.0 mS / cm. In another embodiment, the conductivity of the equilibration buffer, load buffer, or wash buffer is about 10.0 mS / cm to 13.0 mS / cm.

[0183] In one embodiment, the pH of the equilibration buffer, load buffer, or wash buffer is selected from the range of about 6.5 to about 7.5. In a preferred embodiment, the pH of the equilibration buffer is about 7.0.

[0184] In one embodiment, the loading buffer has a concentration range of about 5 mM to about 40 mM. In one embodiment, the loading buffer has a concentration range of about 10 mM to about 30 mM. In a preferred embodiment, the loading buffer has a concentration of about 20 mM.

[0185] In certain embodiments, the affinity chromatography has at least one wash buffer, hi other embodiments, the affinity chromatography has three wash buffers.

[0186] In one embodiment, the first wash buffer has a concentration range of about 5 mM to about 40 mM. In a specific embodiment, the first wash buffer has a concentration range of about 10 mM to about 25 mM. In a preferred embodiment, the concentration of the first wash buffer is about 20 mM.

[0187] In one embodiment, the second wash buffer is selected from sodium phosphate, Tris-HCl, Tris-acetate, HEPES, and glycine-NaOH.

[0188] In certain embodiments, the second wash buffer is used in combination with a salt.

[0189] In certain embodiments, the salt is selected from sodium chloride and potassium chloride, hi a preferred embodiment, the salt is sodium chloride.

[0190] In one embodiment, the second wash buffer has a concentration ranging from about 5 mM to about 40 mM. In a specific embodiment, the second wash buffer has a concentration ranging from about 10 mM to about 25 mM. In a preferred embodiment, the concentration of the second wash buffer is about 20 mM.

[0191] In one embodiment, the second wash 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 wash buffer is about 1.0 M.

[0192] In one embodiment, the second wash buffer has a conductivity range of about 70 mS / cm to about 120 mS / cm. In one embodiment, the second wash buffer has a conductivity range of about 80 mS / cm to about 100 mS / cm. In a preferred embodiment, the conductivity of the second wash buffer is about 90 mS / cm.

[0193] In one embodiment, the pH of the second wash buffer is selected from the range of about 6.5 to about 7.5, and in a preferred embodiment, the pH of the second wash buffer is about 7.0.

[0194] In one embodiment, the second wash buffer further comprises a surfactant selected from polysorbate 20, polysorbate 80, and Triton X-100. In one embodiment, the preferred surfactant is polysorbate 20.

[0195] In one embodiment, the proportion of the surfactant in the second wash buffer is about 0.01% to about 1%. In a preferred embodiment, the proportion of the surfactant in the second wash buffer is 0.1% (w / v).

[0196] In one embodiment, the third wash buffer has a concentration ranging from about 5 mM to about 40 mM. In a specific embodiment, the third wash buffer has a concentration ranging from about 10 mM to about 30 mM. In a preferred embodiment, the concentration of the third wash buffer is about 20 mM.

[0197] In one embodiment, the third wash buffer has a concentration ranging from about 5 mM to about 40 mM. In a specific embodiment, the third wash buffer has a concentration ranging from about 10 mM to about 40 mM. In a preferred embodiment, the concentration of the third wash buffer is about 30 mM.

[0198] In one embodiment, the third wash 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 wash buffer is less than 2.5 mS / cm.

[0199] In one embodiment, the pH of the third wash buffer is selected from about 5 to about 6. In a preferred embodiment, the pH of the third wash buffer is about 5.5.

[0200] In one embodiment, the elution buffer is selected from acetic acid, phosphoric acid, sodium acetate, and HCl. In a preferred embodiment, the elution buffer is acetic acid.

[0201] In one embodiment, the elution buffer is selected from acetic acid, phosphoric acid, sodium acetate, and HCl. In a preferred embodiment, the elution buffer is sodium acetate.

[0202] In one embodiment, the elution buffer has a concentration range selected from about 25 mM to about 250 mM.

[0203] In one embodiment, the elution buffer has a concentration range selected from about 100 mM to about 250 mM. In one embodiment, the elution buffer has a concentration range selected from about 125 mM to 200 mM.

[0204] In one embodiment, the elution buffer has a conductivity range of about 0.2 mS / cm to about 1.0 mS / cm. In one embodiment, the elution buffer has a conductivity range of about 0.5 mS / cm to about 1.0 mS / cm. In one embodiment, the elution buffer has a conductivity range of about 0.5 mS / cm to about 0.6 mS / cm. In one embodiment, the elution buffer has a conductivity range of about 0.2 mS / cm to about 0.3 mS / cm.

[0205] In one embodiment, the pH of the elution buffer is selected from the range of 2.5 to about 3.5. In a preferred embodiment, the pH of the elution buffer is about 3.5.

[0206] In one embodiment, the elution buffer has a conductivity range of about 0.2 mS / cm to about 0.3 mS / cm. 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.

[0207] In certain embodiments, the elution is performed with a linear gradient. In certain embodiments, the elution is performed with a step gradient.

[0208] In one embodiment, collection of the elution peak begins at about 2.5 AU / cm upper value and ends at about 2.5 AU / cm lower value.

[0209] In one embodiment, collection of the elution peak begins at about 0.25 AU / cm upper value and ends at about 0.25 AU / cm lower value.

[0210] In one embodiment, the 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, or less than about 10 NTU, obtained from affinity chromatography, wherein the elution buffer has a concentration of about 200 mM.

[0211] In another embodiment, the present invention provides a process for purifying antibodies or fragments thereof by using affinity chromatography with low salt elution.

[0212] In another embodiment, the present invention provides a process for purifying antibodies or fragments thereof by using affinity chromatography with low salt elution, whereby the eluted protein mixture does not become less turbid during viral inactivation compared to elution performed at high salt concentrations.

[0213] In one embodiment, the equilibration is performed for about 3 CV to about 10 CV. In a preferred embodiment, the equilibration is performed for about 5 CV. In one embodiment, the equilibration is performed until the conductivity endpoint of the equilibration buffer is reached.

[0214] In one embodiment, the amount of protein loaded onto the column during loading is about 10 g / L to about 45 g / L. In another embodiment, the amount of protein loaded onto the column during loading is about 10 g / L to about 50 g / L.

[0215] In one embodiment, the first wash is carried out for at least 1 to about 5 CV. In a preferred embodiment, the first wash is carried out for 3 CV. In one embodiment, the first wash is carried out until the buffer conductivity endpoint is reached.

[0216] In one embodiment, the second wash is carried out for at least 1 CV to about 5 CV. In a preferred embodiment, the second wash is carried out for 3 CV. In one embodiment, the second wash is carried out until the buffer conductivity endpoint is reached.

[0217] In one embodiment, the third wash is carried out for at least 4 CV to about 8 CV. In a preferred embodiment, the third wash is carried out for 5 CV. In one embodiment, the third wash is carried out until the buffer conductivity endpoint is reached.

[0218] In one embodiment, the residence time of the protein in the column during purification with Protein A ranges from about 2 minutes to about 6 minutes. In a preferred embodiment, the residence time of the protein in the column is about 4 minutes.

[0219] AEX chromatography conditions: In one embodiment, the anion exchange chromatography resin is selected from Capto Q, DEAE Sepharose fast flow, Fractogel EMD DEAE(M), Toyopearl DEAE-650, Q Sepharose Fast Flow, POROS XQ, POROS 50 HQ, POROS 50 PI, and POROS 50 D. In a particular embodiment, the anion exchange chromatography resin is strong anion exchange POROS 50 HQ.

[0220] In one embodiment, the equilibration buffer or loading buffer is selected from sodium phosphate, Tris-HCl, HEPES, glycine-NaOH, and Tris-acetate, hi certain embodiments, the equilibration buffer or loading buffer is Tris-acetate or Tris-HCl.

[0221] In one embodiment, the equilibration buffer has a concentration ranging from about 40 mM to about 60 mM. In a specific embodiment, the load buffer has a concentration of about 50 mM. In a specific embodiment, the load buffer has a concentration of about 20 mM.

[0222] In one embodiment, the equilibration buffer has a conductivity range of about 1.5 mS / cm to about 3.5 mS / cm. In a specific embodiment, the conductivity of the equilibration buffer is less than 2.6 mS / cm.

[0223] In one embodiment, the pH of the equilibration buffer is selected from about 6.5 to about 7.5. In a specific embodiment, the pH of the load buffer is about 7.0 to about 7.5. In one embodiment, the pH of the load buffer is 7.2 to 7.4.

[0224] In certain embodiments, the conductivity of the equilibration buffer is ≦2.0 mS / cm.

[0225] In one embodiment, the pH of the equilibration buffer is selected from the range of about 6.5 to about 7.5.

[0226] In certain embodiments, the pH of the load buffer is about 7.0 to about 7.5, hi one embodiment, the pH of the load buffer is 7.2 to 7.3.

[0227] In one embodiment, the loading buffer has a concentration range of about 40 mM to about 60 mM, hi a specific embodiment, the loading buffer has a concentration of about 50 mM.

[0228] In one embodiment, the loading buffer has a concentration range of about 10 mM to about 30 mM, hi a specific embodiment, the loading buffer has a concentration of about 20 mM.

[0229] In one embodiment, the load buffer has a conductivity range of about 1.5 mS / cm to about 3.5 mS / cm, hi a specific embodiment, the conductivity of the load buffer is less than 2.6 mS / cm.

[0230] In one embodiment, the pH of the load buffer is selected from about 6.5 to about 7.5.

[0231] In certain embodiments, the pH of the load buffer is about 7.0 to about 7.5, hi one embodiment, the pH of the load buffer is 7.2 to 7.3.

[0232] In certain embodiments, the conductivity of the load buffer is about ≦3.0 mS / cm.

[0233] In one embodiment, the pH of the load buffer is selected from about 6.5 to about 7.5.

[0234] In certain embodiments, the pH of the load buffer is about 7.0 to about 7.5, hi one embodiment, the pH of the load buffer is 7.2 to 7.3.

[0235] In one embodiment, the present invention provides protein peak recovery criteria selected from terminating at about 2.5 AU / cm upper value and about 1.5 AU / cm lower value.

[0236] In one embodiment, the present invention provides a protein peak recovery criterion selected from terminating at about 1.5 AU / cm upper value and about 1.5 AU / cm lower value.

[0237] In one embodiment, the present invention provides an antibody composition comprising an antibody of interest obtained from AEX chromatography with a peak recovery standard selected from about 2.5 AU / cm to about 1.5 AU / cm, and about 10% to 12% acidic variants.

[0238] In another embodiment, the present invention provides protein peak recovery criteria selected from terminating at about 1.5 AU / cm upper value and about 1.5 AU / cm lower value.

[0239] In one embodiment, the wash buffer is selected from sodium phosphate, Tris-HCl, HEPES, glycine-NaOH, and Tris-acetate.

[0240] In one embodiment, the wash buffer has a concentration ranging from about 40 mM to about 60 mM. In a specific embodiment, the wash buffer has a concentration of about 50 mM.

[0241] In another embodiment, the wash buffer has a concentration range of about 10 mM to about 30 mM. In a specific embodiment, the concentration of the wash buffer is about 20 mM.

[0242] In one embodiment, the wash buffer has a conductivity range of about 1.5 mS / cm to about 3.5 mS / cm. In a preferred embodiment, the wash buffer has a conductivity of less than 2.6 mS / cm.

[0243] In certain embodiments, the conductivity of the wash buffer is ≦2.0 mS / cm.

[0244] In one embodiment, the pH of the wash buffer is selected from about 6.5 to about 7.5, and in a specific embodiment, the pH of the wash buffer is about 7.2.

[0245] In one embodiment, the refolding buffer is selected from sodium phosphate, Tris-HCl, HEPES, glycine-NaOH, and Tris-acetate.

[0246] In one embodiment, the renaturation buffer has a concentration range of about 5 mM to about 30 mM, hi a specific embodiment, the concentration of the renaturation buffer is about 20 mM.

[0247] In one embodiment, the regeneration buffer also comprises a salt selected from sodium chloride, potassium chloride, and calcium chloride. In a particular embodiment, the salt in the regeneration buffer is sodium chloride.

[0248] In one embodiment, the salt in the renaturation buffer has a concentration ranging from about 0.5 M to about 1.5 M. In a specific embodiment, the salt in the renaturation buffer has a concentration of about 1.0 M.

[0249] In one embodiment, the regeneration buffer has a conductivity range of about 80 mS / cm to about 90 mS / cm. In a specific embodiment, the conductivity of the regeneration buffer is about 85 mS / cm.

[0250] In one embodiment, the regeneration buffer has a conductivity range of about 90 mS / cm to about 110 mS / cm. In a specific embodiment, the regeneration buffer has a conductivity of about 100 mS / cm.

[0251] In one embodiment, the pH of the refolding buffer is selected from the range of about 6.5 to about 7.5. In a preferred embodiment, the pH of the refolding buffer is about 7.0.

[0252] In one embodiment, the pH of the renaturation buffer is selected from about 6.5 to about 7.5, and in a preferred embodiment, the pH of the renaturation buffer is about 7.2.

[0253] In one embodiment, the elution is performed in flow-through mode.

[0254] In one embodiment, the sanitization buffer is selected from NaOH, isopropyl alcohol, and benzyl alcohol. In a specific embodiment, the sanitization buffer is NaOH.

[0255] In one embodiment, the sanitization buffer has a concentration range of about 300 mM to about 1500 mM. In a specific embodiment, the concentration of the renaturation buffer is about 500 mM.

[0256] In one embodiment, the loading is carried out at least about 5 CV or more, hi a specific embodiment, the loading is carried out at about 30 CV.

[0257] In one embodiment, the equilibration is carried out for at least about 3 CV to about 10 CV, hi a specific embodiment, the equilibration is carried out for about 5 CV.

[0258] In one embodiment, the equilibration is carried out until the endpoint of the conductivity of the equilibration buffer is reached.

[0259] In one embodiment, the amount of protein loaded onto the column at the time of loading is selected from less than about 150 g / L, less than about 130 g / L, less than about 120 g / L, less than about 110 g / L, or less than about 100 g / L.

[0260] In one embodiment, the wash is carried out for at least about 5 CV.

[0261] In one embodiment, the wash is carried out for at least about 2 CV.

[0262] In one embodiment, the regeneration is carried out for at least 2 CV to about 5 CV, hi a particular embodiment, the regeneration is carried out for about 3 CV.

[0263] In one embodiment, the regeneration removes most of the impurities. In a preferred embodiment, the regeneration removes most of the HMW and acidic charge variant based impurities.

[0264] In one embodiment, the sanitization is carried out for at least 2 CV to about 5 CV, hi a specific embodiment, the sanitization is carried out for about 3 CV.

[0265] In one embodiment, the sanitization buffer is retained in the column for about 15 minutes to about 60 minutes, hi a specific embodiment, the sanitization buffer is retained in the column for about 20 minutes.

[0266] In one embodiment, the retention time of the protein in the column during AEX purification ranges from about 2 minutes to about 6 minutes, and in a specific embodiment, the retention time of the protein in the column is about 4 minutes.

[0267] Ceramic Hydroxyapatite (CHT) Conditions: In one embodiment, the ceramic hydroxyapatite is CHT (ceramic hydroxyapatite)-Type 1 and CHT (ceramic hydroxyapatite) XT.

[0268] In one embodiment, Buffer A and Buffer B are combined to form the load buffer, wash buffer, equilibration buffer, and elution buffer.

[0269] In certain embodiments, Buffer A is a polar protic molecule selected from water (H—OH), acetic acid (CHCO—OH), methanol (CH—OH), ethanol (CHCH—OH), n-propanol (CHCHCH—OH), and n-butanol (CHCHCHCH—OH).

[0270] In a preferred embodiment, the polar proton molecule is water.

[0271] In one embodiment, buffer B is selected from sodium phosphate, Tris, HEPES, glycine-NaOH, In a preferred embodiment, said buffer B is sodium phosphate.

[0272] In one embodiment, the concentration range of Buffer B is about 20 mM to about 40 mM. In a specific embodiment, the concentration of Buffer B is about 32 mM.

[0273] In one embodiment, the concentration range of Buffer B is about 20 mM to about 40 mM. In a specific embodiment, the concentration of Buffer B is about 40 mM.

[0274] In one embodiment, the concentration range of the equilibration buffer and the load buffer is about 20 mM to about 40 mM, and in a specific embodiment, the concentration of the equilibration buffer and the load buffer is about 32 mM.

[0275] In certain embodiments, the strengths of the equilibration buffer, load buffer, and wash buffer are the same. In one embodiment, the strength of the elution buffer is higher than the load buffer.

[0276] In one embodiment, the equilibration buffer and the load buffer have a concentration range of about 20 to about 50 mM. In a specific embodiment, the concentration of the equilibration buffer is about 40 mM.

[0277] In one embodiment, the equilibration buffer has a concentration of about 20 mM to 50 mM. In a specific embodiment, the equilibration buffer has a concentration of about 32 mM. In one embodiment, the pH of the equilibration buffer, load buffer, wash buffer, or elution buffer is selected from about 6.5 to about 7.5.

[0278] In one embodiment, the pH of the buffer is about 7.0±0.2.

[0279] In one embodiment, the equilibration buffer has a conductivity range of about 3.0 to about 7.0 mS / cm. In a preferred embodiment, the conductivity of the equilibration buffer is about 6.0 mS / cm.

[0280] In one embodiment, the pH of the equilibration buffer is selected from the range of about 6.5 to about 7.5.

[0281] In one embodiment, the pH of the equilibration buffer is about 7.0±0.2.

[0282] In one embodiment, the equilibration is performed with an isocratic gradient by using a combination of at least two buffers, where the concentration of buffer A is at least 10% of the concentration of buffer B. In a preferred embodiment, the concentration of buffer A is at least 8% of the concentration of buffer B.

[0283] In one embodiment, the loading buffer has a concentration range of about 30 to about 60 mM. In a preferred embodiment, the loading buffer is about 40 mM.

[0284] In one embodiment, the loading buffer has a concentration of about 10 mM to 40 mM, hi a specific embodiment, the loading buffer has a concentration of about 32 mM.

[0285] In one embodiment, the load buffer has a conductivity range of about 3.0 to about 7.0 mS / cm. In a preferred embodiment, the load buffer has a conductivity of about 6.0 mS / cm.

[0286] In one embodiment, the pH of the load buffer is selected from about 6.5 to about 7.5.

[0287] In one embodiment, the pH of the loading buffer is about 7.0±0.2.

[0288] In one embodiment, the loading is performed in an isocratic gradient by using a combination of at least two buffers, where the concentration of buffer A is at least 10% of the concentration of buffer B. In a preferred embodiment, the concentration of buffer A is at least 8% of that of buffer B.

[0289] In one embodiment, the wash buffer has a concentration ranging from about 30 mM to about 60 mM. In a preferred embodiment, the wash buffer has a concentration of about 40 mM.

[0290] In one embodiment, the wash buffer has a concentration of about 10 mM to 40 mM, hi a specific embodiment, the wash buffer has a concentration of about 32 mM.

[0291] In one embodiment, the wash buffer has a conductivity range of about 3.0 mS / cm to about 7.0 mS / cm. In a preferred embodiment, the wash buffer has a conductivity of about 6.0 mS / cm.

[0292] In one embodiment, the pH of the wash buffer is selected from about 6.5 to about 7.5.

[0293] In one embodiment, the pH of the wash buffer is about 7.0±0.2.

[0294] In one embodiment, the washing is performed in an isocratic gradient by using a combination of at least two buffers, where the concentration of buffer A is at least 10% or less than that of buffer B. In a preferred embodiment, the concentration of buffer A is at least 10% of the concentration of buffer B.

[0295] In one embodiment, the elution buffer has a concentration range of about 10 mM to about 400 mM. In a preferred embodiment, the elution buffer has a concentration range of about 32 mM to about 104 mM.

[0296] In one embodiment, the elution buffer has a concentration range of about 10 mM to about 400 mM. In a preferred embodiment, the elution buffer has a concentration range of about 24 mM to about 100 mM.

[0297] In one embodiment, the elution buffer has a concentration range of about 10 mM to about 400 mM, and in a preferred embodiment, the elution buffer has a concentration range of about 32 mM to about 88 mM.

[0298] In one embodiment, the elution buffer has a concentration range of about 10 mM to about 400 mM. In a preferred embodiment, the elution buffer has a concentration range of about 40 mM to about 96 mM.

[0299] In one embodiment, the elution buffer has a conductivity range of about 6 mS / cm to about 12 mS / cm.

[0300] In one embodiment, the pH of the elution buffer is selected from about 6.5 to about 7.5, In one embodiment, the pH of the elution buffer is about 7.0±0.2.

[0301] In one embodiment, the elution is performed with a linear gradient. In a particular embodiment, the elution is performed with a step gradient.

[0302] In one embodiment, the elution is performed in a linear gradient by using a combination of at least two buffers, where the concentration of buffer A is at least 10% of the concentration of buffer B. In such an embodiment, the concentration of buffer A is at least 10% of the concentration of buffer B. In such an embodiment, buffer B is gradually increased from about 10% to about 24%, and the concentration of buffer B is optimized accordingly.

[0303] Furthermore, one skilled in the art will appreciate that the slope will vary slightly with run time and the particular impurity being removed.

[0304] In such embodiments, elution is performed with an isocratic gradient at 15% Buffer B. In such embodiments, elution is performed with an isocratic gradient at 20% Buffer B. In such embodiments, elution is performed with an isocratic gradient at 15%, 16%, 17%, 18%, 19%, and 20% Buffer B.

[0305] In another embodiment, the elution is performed in a linear gradient by using a combination of at least two buffers, where the concentration of buffer A is at least 8% of the concentration of buffer B. In such an embodiment, the concentration of buffer A is at least 8% of the concentration of buffer B. In such an embodiment, buffer B is gradually increased from about 8% to about 22%, and the concentration of buffer B is optimized accordingly.

[0306] In one embodiment, to minimize LMW, collection of the elution peak begins at about 1.5 AU / cm upper value and ends at about 2.0 AU / cm lower value.

[0307] In one embodiment, to minimize LMW, collection of the elution peak begins at about 1.0 AU / cm upper value and ends at about 1.5 AU / cm lower value.

[0308] In certain embodiments, the CHT is performed in bind and elute mode.

[0309] In certain embodiments, the CHT is performed in a buffer or solvent that does not contain calcium chloride.

[0310] In certain embodiments, the concentration of the elution buffer is higher than that of the wash buffer.

[0311] In one embodiment, the elution buffer comprises at least 8% of a 400 mM salt buffer concentration. In one embodiment, the elution buffer comprises at least 10% of a 400 mM salt buffer concentration. In one embodiment, the elution buffer comprises at least 15% of a 400 mM salt buffer concentration. In one embodiment, the elution buffer comprises at least 20% of a 400 mM salt buffer concentration. In one embodiment, the elution buffer comprises at least 26% of a 400 mM salt buffer concentration.

[0312] In one embodiment, the elution is carried out at about 5 CV to about 10 CV.

[0313] In one embodiment, the elution is carried out at about 10 CV to about 15 CV.

[0314] In one embodiment, the elution is carried out at about 15 to about 20 CV or more, and in a preferred embodiment, the elution is carried out at about 19 CV.

[0315] In one embodiment, the equilibration is carried out at about 3 to about 10 CV, and in a preferred embodiment, the equilibration is carried out at about 5 CV.

[0316] In one embodiment, the equilibration is carried out until the endpoint of the conductivity of the equilibration buffer is reached.

[0317] In one embodiment, the amount of protein loaded onto the column during loading ranges from about 5 g / L to about 25 g / L, and in one embodiment, the amount of protein loaded onto the column during loading is 12.5 g / L.

[0318] In one embodiment, the wash is performed for about 2 CV to about 5 CV. In a preferred embodiment, the wash is performed for about 3 CV. In one embodiment, the wash is performed until the endpoint of the conductivity of the buffer is reached.

[0319] In one embodiment, the residence time of the protein in the CHT column during equilibration, washing, and loading ranges from about 2 minutes to about 6 minutes. In a preferred embodiment, the residence time of the protein in the column during equilibration, washing, and loading is about 4 minutes.

[0320] In one embodiment, the residence time of the protein in the column during elution ranges from about 2 minutes to about 6 minutes. In a preferred embodiment, the residence time of the protein in the column during elution is about 4 minutes. [Example]

[0321] The present invention is provided below for illustrative purposes only and should not be construed as being limited to the following examples.

[0322] Example 1 - Purification of monoclonal antibodies by affinity column followed by anion exchange column: All chromatography processes were performed using an AKTA Pure 150 system from GE Healthcare. The concentration of protein samples was determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. Mabselect Sure LX resin media was obtained from GE Healthcare. Vantage and C10 / 20 columns were obtained from Millipore Corporation and GE Healthcare, respectively. Turbidity measurements were performed using columns obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were of GMP grade.

[0323] 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, GE Healthcare) packed onto a VL11 / 250 or C10 / 20 column. The residence time was 4 minutes for all steps. After equilibration with Tris acetate + 100 mM NaCl, pH 6.8-7.2, the clarified harvest was loaded at ≤45 mg / mL of resin. After loading, the column was washed with equilibration buffer (Wash 1), followed by Wash 2 and Wash 3 buffers, as described in Table 1. The affinity chromatography step was performed in bind and elute mode, with collection from 50 mAU (0.25 AU / cm) above the peak to 50 mAU (0.25 AU / cm) below. The Protein A output was analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the Protein A step is summarized in Table 1. Table 1: Experimental design for Protein A [Table 1]

[0324] The eluted protein is further subjected to viral inactivation and neutralization. After neutralization, the protein is filtered through a 0.2 μm filter.

[0325] After the virus inactivation and neutralization steps, the eluted protein mixture is further purified using anion exchange chromatography resin (Poros HQ, Thermofisher) packed in a C10 / 20 column.

[0326] The residence time is 4 minutes for all steps. After equilibration with Tris acetate, pH 7.0-7.3, the neutralized Protein A output is loaded at ≦100 mg / mL of resin. The load is diluted with water to meet the (mS / cm) conductivity specification, i.e., ≦3.0 mS / cm, before application to the AEX column.

[0327] The AEX step was performed in flow-through (negative) mode, collecting from 500 mAU above the peak to 300 mAU below. The column was washed with Tris acetate, pH 7.0-7.3. The AEX output was analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the POROS 50 HQ step is summarized in Table 2. Table 2: Experimental design (AEX) for POROS 50 HQ [Table 2] Table 3: SE (size exclusion)-HPLC (high performance liquid chromatography) analysis [Table 3]

[0328] Table 3 shows a 99% purity of the main peak and a 98% reduction in HMW as determined by SE-HPLC.

[0329] Table 4 shows the results of cation exchange-high performance liquid chromatography comparing the main peak with the acidic variant. [Table 4]

[0330] Table 4 shows a 74% purity of the main peak and approximately 55% reduction of the acidic variant as determined by CEX-HPLC.

[0331] Example 2 - Purification of monoclonal antibodies by affinity chromatography followed by AEX chromatography followed by CHT Type 1 resin: All chromatography processes were performed using an AKTA Pure 150 system from GE Healthcare. The concentration of protein samples was determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. CHT Type 1 resin media was obtained from Bio-Rad. Vantage and C10 / 20 columns were obtained from Millipore Corporation and GE Healthcare, respectively. Turbidity measurements were performed using columns obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were of GMP grade.

[0332] Monoclonal antibody molecules capable of binding to IgE molecules expressed in Chinese hamster ovary (CHO) cell lines are captured using Protein A (Mab Select Sure LX, GE Healthcare) packed into a VL11 / 250 column. The eluted proteins are then subjected to viral inactivation and neutralization. After neutralization, the proteins are filtered through a 0.2 μm filter.

[0333] After the virus inactivation and neutralization steps, the protein mixture is further purified using anion exchange chromatography resin (Poros HQ, Thermofisher) packed in a C10 / 20 column.

[0334] The protein eluted from anion exchange chromatography (AEX) is further polished using a mixed-mode resin (CHT Type I, Bio-Rad) packed in a VL11 / 250 column. The residence time is 4 min / 3 min for all steps. After equilibration with sodium phosphate, pH 6.8, the anion exchange (AEX) output is loaded onto the resin at 12.5 mg / mL. The column is washed with 3 CV of sodium phosphate, pH 6.8. The bound target protein is eluted using a linear gradient from 32 mM sodium phosphate to 400 mM sodium phosphate, pH 6.8. The eluted peak of interest is analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design and process for the CHT Type I step are summarized in Table 5. Table 5: Experimental design for CHT type I [Table 5]

[0335] Example 3 - Purification of monoclonal antibodies by affinity chromatography followed by AEX chromatography followed by CHT XT resin: All chromatography processes were performed using a GE Healthcare AKTA Pure 150 system. Protein sample concentrations were determined by measuring absorbance at 280 nm using a Shimadzu spectrophotometer. CHT XT resin media was obtained from Bio-Rad. Vantage and C10 / 20 columns were obtained from Millipore Corporation and GE Healthcare, respectively. Turbidity measurements were performed using columns obtained from Thermo Scientific. All chemicals were obtained from JTB or Merck Millipore and were GMP grade.

[0336] Monoclonal antibodies capable of binding to IgE expressed in a Chinese hamster ovary (CHO) cell line are captured using Protein A packed into a VL11 / 250 column (Mab Select Sure LX, GE Healthcare).

[0337] The eluted protein is further subjected to viral inactivation and neutralization. After neutralization, the protein is filtered through a 0.2 μm filter.

[0338] After the virus inactivation and neutralization steps, the eluted protein mixture is further purified using anion exchange chromatography resin (Poros HQ, Thermofisher) packed in a C10 / 20 column.

[0339] The protein eluted from anion exchange chromatography (AEX) was further polished using a mixed-mode resin (CHT XT, Bio-Rad) packed in a VL11 / 250 column. The residence time was 4 min / 3 min for all steps. After equilibration with sodium phosphate, pH 6.8, the anion exchange (AEX) output was loaded onto the resin at 12.5 mg / mL. The column was washed with 3 CV of sodium phosphate, pH 6.8. The bound target protein was eluted using a linear gradient from 40 mM sodium phosphate to 400 mM sodium phosphate, pH 6.8. The eluted peak of interest was analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design and process for the CHT XT step are summarized in Tables 6–9. Table 6: Experimental design for CHT XT [Table 6] Table 7: SE-HPLC analysis of the eluate from the CHT Type I / XT column [Table 7] The SE-HPLC data reveal that the composition obtained from CHT has a significantly reduced LMW (approximately 100%) and a protein purity of 99.99%. Table 8: Size-related variants: CE-SDS [Table 8] From the data it is clear that the composition obtained from CHT has a significantly reduced (about 81%) LMW. Table 9: Charge-related variants: CEX-HPLC [Table 9]

[0340] Table 9 shows a purity of 84% of the main peak and about 56% reduction in LMW as determined by CEX-HPLC.

[0341] Example 4 - Purification of monoclonal antibodies by affinity column followed by anion exchange column (50 L scale) All chromatography processes were performed using GE Healthcare's AKTA Pilot or AKTA Process. Protein sample concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. Mabselect Sure LX resin media was obtained from GE Healthcare. Chromatography 100 / 250 columns were obtained from Millipore Corporation, GE Healthcare, or Repligen. All chemicals used in the manufacturing process were GMP grade.

[0342] Monoclonal antibody molecules capable of binding to IgE molecules expressed in a Chinese hamster ovary (CHO) cell line were captured using Protein A (Mab Select Sure LX, GE Healthcare) packed into a chromatography 100 / 250 column. The residence time was 4 minutes for all steps. After equilibration with Tris-HCl + 150 mM NaCl, pH 6.8-7.2, the clarified harvest was loaded at ≤45 mg / mL on the resin. After loading, the column was washed with equilibration buffer (Wash 1), followed by Wash 2 and Wash 3 buffers, as described in Table 10. The affinity chromatography step was performed in bind and elute mode, with collection from 0.25 AU / cm above to 0.25 AU / cm below the peak. The Protein A output was analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the Protein A step is summarized in Table 10. Table 10: Protein A experimental design [Table 10]

[0343] The eluted protein is further purified using anion exchange chromatography resin (Poros 50 HQ, Thermofisher) packed in a chromatography 100 / 250 column. The residence time is 4 minutes for all steps. After equilibration with Tris-HCl, pH 7.1-7.3, the neutralized Protein A output is loaded at 70-145 mg / mL on the resin. The load is diluted with water to meet the (mS / cm) conductivity specification, i.e., ≤2.0 mS / cm, before application to the AEX column.

[0344] The AEX step is performed in flow-through (negative) mode, with collection from 2.0 AU / cm above the peak to 2.5 AU / cm below. The column is washed with Tris-HCl, pH 7.1-7.3. The AEX output is analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the POROS 50 HQ step is summarized in Table 11. Table 11: Experimental design for AEX [Table 11] Table 12: SE (size exclusion)-HPLC (high performance liquid chromatography) analysis [Table 12]

[0345] Table 12 shows a 99% purity of the main peak and a 96% reduction in HMW as determined by SE-HPLC. Table 13: Cation exchange-high performance liquid chromatography results comparing the main peak with the acidic variant. [Table 13]

[0346] Table 13 shows a 67% purity of the main peak and approximately 25% reduction of the acidic variant as determined by CEX-HPLC.

[0347] Example 5 - Purification of monoclonal antibodies by affinity chromatography followed by AEX chromatography followed by CHT XT resin: All chromatography processes were performed using GE Healthcare's AKTA Pilot or AKTA Process systems. Protein sample concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. CHT XT resin media was obtained from Bio-Rad. Chromatography 100 / 250 columns were obtained from Millipore Corporation, GE Healthcare, or Repligen. All chemicals used in the manufacturing process were GMP grade.

[0348] Monoclonal antibodies capable of binding to IgE expressed in Chinese hamster ovary (CHO) cell lines were captured using Protein A (Mab Select Sure LX, GE Healthcare) packed into a chromatography 100 / 250 column. The eluted proteins were further subjected to viral inactivation and neutralization, followed by depth filtration. After depth filtration, the proteins were filtered through a 0.2 μm filter.

[0349] After the depth filtration step, the protein mixture is further purified using an anion exchange chromatography resin (Poros HQ, Thermofisher) packed in a chromatography 100 / 250 column.

[0350] The protein eluted from anion exchange chromatography (AEX) is further polished using a mixed-mode resin (CHT XT, Bio-Rad) packed in a 100 / 250 column. The residence time is 4 min / 5 min for all steps. After equilibration with sodium phosphate, pH 6.8, the anion exchange (AEX) output is loaded at 12.5 mg / mL on the resin. The column is washed with 3 CV of sodium phosphate, pH 6.8. The bound target protein is eluted using a linear gradient from 40 mM sodium phosphate to 400 mM sodium phosphate, pH 6.8. The eluted peak of interest is analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design and process for the CHT XT step are summarized in Tables 14-17. Table 14: Experimental design for CHT XT [Table 14] Table 15: SE HPLC analysis of LMW [Table 15] Table 16: CEX HPLC-BV analysis [Table 16] Table 17: Analysis of LMW by CE-SDS [Table 17] Results: The table shows that the LMW was reduced by a total of 40%.

[0351] Example 6 - Purification of monoclonal antibodies by affinity column followed by anion exchange column (200 L scale) All chromatography processes were performed using GE Healthcare's AKTA Pilot or AKTA Process. Protein sample concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. Mabselect Sure LX resin media was obtained from GE Healthcare. Chromatography 250 / 250 columns were obtained from Millipore Corporation, GE Healthcare, or Repligen. All chemicals used in the manufacturing process were GMP grade.

[0352] Monoclonal antibody molecules capable of binding to IgE molecules expressed in a Chinese hamster ovary (CHO) cell line were captured using Protein A (Mab Select Sure LX, GE Healthcare) packed into a chromatography 250 / 250 column. The residence time was 4 minutes for all steps. After equilibration with Tris-HCl + 150 mM NaCl, pH 6.8-7.2, the clarified harvest was loaded at ≤45 mg / mL on the resin. After loading, the column was washed with equilibration buffer (Wash 1), followed by Wash 2 and Wash 3 buffers, as described in Table 18. The affinity chromatography step was performed in bind and elute mode, with collection from 0.25 AU / cm above to 0.25 AU / cm below the peak. The Protein A output was analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the Protein A step is summarized in Table 18. Table 18: Protein A experimental design [Table 18]

[0353] The eluted protein is further purified using anion exchange chromatography resin (POROS 50 HQ, Thermofisher) packed in a 350 / 250 or 200 / 350 column. The residence time is 4 minutes for all steps. After equilibration with Tris-HCl, pH 7.1-7.3, the neutralized Protein A output is loaded at 70-145 mg / mL on the resin. The load is diluted with water to meet the (mS / cm) conductivity specification, i.e., ≤2.0 mS / cm, before application to the AEX column.

[0354] The AEX step is performed in flow-through (negative) mode, with collection from 2.0 AU / cm above the peak to 2.5 AU / cm below. The column is washed with Tris-HCl, pH 7.1-7.3. The AEX output is analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design for the POROS 50 HQ step is summarized in Table 19. Table 19: Experimental design for AEX [Table 19] Table 20: SE (size exclusion)-HPLC (high performance liquid chromatography analysis) [Table 20]

[0355] Table 20 shows a 99% purity of the main peak and a 91% reduction in HMW as determined by SE-HPLC. Table 21: Cation exchange-high performance liquid chromatography results comparing the main peak with the acidic variant. [Table 21]

[0356] Table 21 shows a 74% purity of the main peak and approximately 46% reduction of the acidic variant as determined by CEX-HPLC.

[0357] Example 7 - Purification of monoclonal antibody by affinity chromatography followed by AEX chromatography followed by CHT XT resin (200 L scale): All chromatography processes were performed using GE Healthcare's AKTA Pilot or AKTA Process systems. Protein sample concentrations were determined by measuring absorbance at 280 nm using a spectrophotometer. CHT XT resin media was obtained from Bio-Rad. Chromatography 300 / 250 columns were obtained from Millipore Corporation, GE Healthcare, or Repligen. All chemicals used in the manufacturing process were GMP grade.

[0358] Monoclonal antibodies capable of binding to IgE expressed in Chinese hamster ovary (CHO) cell lines were captured using Protein A (Mab Select Sure LX, GE Healthcare) packed into a 250 / 250 chromatography column. The eluted proteins were further subjected to viral inactivation and neutralization, followed by depth filtration. After depth filtration, the proteins were filtered through a 0.2 μm filter.

[0359] After the depth filtration step, the protein mixture is further purified using anion exchange chromatography resin (Poros HQ, Thermofisher) packed in a chromatography 350 / 250 or 200 / 250 column.

[0360] The protein eluted from anion exchange chromatography (AEX) is further polished using a mixed-mode resin (CHT XT, Bio-Rad) packed in a 300 / 250 column. The residence time is 4 min / 5 min for all steps. After equilibration with sodium phosphate, pH 6.8, the anion exchange (AEX) output is loaded onto the resin at a maximum concentration of 12.5 mg / mL. The column is washed with 3 CV of sodium phosphate, pH 6.8. The bound target protein is eluted using a linear gradient from 40 mM sodium phosphate to 400 mM sodium phosphate, pH 6.8. The eluted peak of interest is analyzed for size and charge variants using SE-HPLC and CEX-HPLC. The experimental design and process for the CHT XT step are summarized in Tables 22–24. Table 22: Experimental design for CHT XT [Table 22] Table 23: SE HPLC analysis of LMW [Table 23] Table 24: CEX HPLC-BV analysis [Table 24]

Claims

1. 1. A process for purifying a protein of interest from a protein mixture comprising said protein of interest and one or more low molecular weight (LMW) variants, comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. Eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The process wherein the protein of interest is substantially pure and has less than 0.4% LMW when analyzed by SE-HPLC analysis.

2. 1. A process for purifying a protein of interest from a protein mixture comprising said protein of interest, one or more low molecular weight (LMW) variants, and one or more basic variants (BV), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. Eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

3. 3. The process of claim 1 or claim 2, wherein the purified protein of interest contains at least 97% reduced LMW as measured by SE-HPLC analysis.

4. 3. The process of claim 1 or claim 2, wherein the purified protein of interest contains at least 95% reduced LMW as measured by SE-HPLC analysis.

5. 3. The process of claim 1 or claim 2, wherein the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

6. 3. The process of claim 1 or claim 2, wherein the protein mixture eluted from the CHT column contains less than 0.4% LMW when analyzed by SE-HPLC analysis.

7. 3. The process of claim 1 or claim 2, wherein the LMW is selected from LC, HC, HH, and 2H1L.

8. 8. The process of claim 7, wherein the amount of 2H1L is selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, or 1.4% or less as measured by CE-SDS.

9. 8. The process of claim 7, wherein the amount of HH is selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less, or 0.08% or less, 0.06% or less, or 0.04% or less, and 0.03% or less as measured by CE-SDS.

10. 8. The process of claim 7, wherein the amount of HC is selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less, and 0.18% or less as measured by CE-SDS.

11. 8. The process of claim 7, wherein the amount of LC is selected from about 0.5% or less, 0.4% or less, 0.3% or less, and 0.2% or less as measured by CE-SDS.

12. 8. The process of claim 7, wherein the LC, HC, HH, and 2H1L are present in an amount of about 2.2% to about 2.6% as measured by CE-SDS.

13. 1. A process for purifying a protein of interest from a protein mixture comprising the protein of interest and one or more basic variants (BVs), comprising: a. loading the protein mixture onto a ceramic hydroxyapatite (CHT) column in an appropriate buffer; b. washing the CHT column with an appropriate buffer; c. Eluting the purified protein of interest with an appropriate gradient of phosphate buffer; Including, The purified protein of interest is substantially pure and contains less than 15% of the basic variant when analyzed by CEX-HPLC analysis.

14. 14. The process of claim 2 or claim 13, wherein the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

15. 15. The process of claim 14, wherein the protein mixture eluted from the CHT column contains less than 10% BV.

16. 15. The process of claim 14, wherein the protein mixture eluted from the CHT column comprises 5% BV.

17. 14. The process of claim 1, claim 2, or claim 13, wherein the elution phosphate gradient is a linear gradient that gradually increases the concentration of phosphate from about 24 mM to 104 mM.

18. 18. The process of claim 17, wherein the concentration of phosphate is gradually increased from about 40 mM to 96 mM.

19. 18. The process of claim 17, wherein the concentration of phosphate is gradually increased from about 32 mM to 88 mM.

20. 18. The process of claim 17, wherein the elution phosphate gradient is a linear gradient in which the concentration of phosphate gradually increases from about 5% to 25% buffer B.

21. 21. The process of claim 20, wherein the elution phosphate gradient is a linear gradient in which the concentration of phosphate gradually increases from about 10% to 24% buffer B.

22. 22. The process of claim 21, wherein the concentration of phosphate is gradually increased from about 8% to 22% in buffer B.

23. 14. The process of claim 1, claim 2, or claim 13, wherein gradient elution is performed over 19 CV.

24. 14. The process of claim 1, claim 2, or claim 13, wherein the elution begins at an upper value of about 1.0 AU / cm and ends at a lower value of about 1.5 AU / cm.

25. 14. The process of claim 1, claim 2, or claim 13, wherein the CHT column is treated with an equilibration buffer prior to treatment with a load buffer.

26. 14. The process of claim 1, claim 2, or claim 13, wherein the load and wash buffers have the same conductivity and pH.

27. 14. The process of claim 1, claim 2, or claim 13, wherein the load, wash, and elution buffers have a pH of 7.0±0.

2.

28. 14. The process of claim 1, claim 2, or claim 13, wherein the load, wash, and elution buffers do not contain an additive selected from sodium chloride, calcium chloride, and glycine.

29. 14. The process of claim 1, claim 2, or claim 13, wherein the CHT column is performed after Protein A chromatography.

30. 14. The process of claim 1, claim 2, or claim 13, wherein the CHT column is followed by anion exchange chromatography.

31. 1. A process for purifying a protein of interest from a protein mixture, comprising: a. obtaining a protein mixture from a mammalian expression system containing the protein of interest and at least one impurity selected from an acidic variant, a basic variant, a low molecular weight (LMW), and a high molecular weight (HMW); b. applying the protein mixture to an affinity chromatography column; c. Eluting the protein mixture from the affinity chromatography column; d. performing viral inactivation of the protein mixture obtained in step; e. applying the protein mixture obtained in step (d) to anion exchange chromatography; f. Eluting the protein mixture in a flow-through mode; g. applying the protein mixture obtained in step (f) to a ceramic hydroxyapatite (CHT) column; h. Optionally, washing the CHT column with an appropriate wash buffer; i. Eluting the protein mixture from the CHT column with an appropriate buffer; Including, The process wherein the eluted protein mixture is enriched with the protein of interest and substantially reduced in impurities selected from acidic variants, basic variants, low molecular weight (LMW), and high molecular weight (HMW).

32. 32. The process of claim 31 , wherein the protein mixture eluted from the CHT column comprises an LMW selected from about 0.3% or less LMW, 0.2% or less LMW, or 0.1% or less LMW when analyzed by SE-HPLC analysis.

33. 33. The process of claim 32, wherein the protein mixture eluted from the CHT column contains less than 0.4% LMW when analyzed by SE-HPLC analysis.

34. 32. The process of claim 31 , wherein the protein mixture eluted from the CHT column comprises a low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, and 1% or less BV.

35. 35. The process of claim 34, wherein the protein mixture eluted from the CHT column contains less than 10% BV.

36. 36. The process of claim 35, wherein the protein mixture eluted from the CHT column comprises 5% BV.

37. 32. The process of claim 31, wherein acidic variants are substantially removed by anion exchange chromatography.

38. 38. The process of claim 37, wherein the acidic variants are about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, less than 1% AV.

39. 32. The process of claim 31, wherein high molecular weight impurities (HMW) are substantially removed by anion exchange chromatography.

40. 40. The process of claim 39, wherein the high molecular weight impurities are selected from about 0.5% or less, about 0.4% or less, or 0.3% or less, or 0.2% or less, or 0.1% or less.

41. 41. The process of any one of claims 1 to 40, wherein the protein of interest is selected from an IgG1 antibody or a fragment thereof, and a fusion protein.

42. 42. The process of claim 41, wherein the IgG1 antibody or fusion protein has an isoelectric point of 6 to 9.

43. The IgG1 antibody or fusion protein is selected from the group consisting of 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, raxibacumab, or 42. The process of claim 41, wherein the therapeutic agent is selected from binutuzumab, siltuximab, ramucirumab, vedolizumab, nivolumab, pembrolizumab, dalucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, sarilumab, ocrelizumab, tildrakizumab, romosozumab, brolucizumab, and crizanlizumab.

44. 42. The process of claim 41, wherein the IgG1 antibody binds to IgE.

45. 45. The process of claim 44, wherein the binding IgG1 antibody is omalizumab.

46. 1. A pharmaceutical composition of an antibody that binds to IgE, comprising a substantially purified monomer of said antibody having a purity of at least 90% and less than about 0.2% LMW as measured by SE-HPLC.

47. 1. A pharmaceutical composition of omalizumab, comprising a substantially purified monomer of said omalizumab having a purity of at least 90% and less than about 5% LMW as measured by SE-HPLC.

48. 48. The pharmaceutical composition of claim 46 or claim 47, wherein the purity of the monomer is selected from greater than 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% purity.

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