method for purifying a polypeptide comprising a ch2 / ch3 region

专利摘要:
METHOD FOR PURIFYING A POLYPEPTIDE UNDERSTANDING A CH2 / CH3 REGION. The present invention relates to methods for the purification of a polypeptide comprising a CH2 / CH3 region, comprising binding the polypeptide to Protein A and eluting with a pH gradient starting at a low pH.
公开号:BR112012004697B1
申请号:R112012004697-7
申请日:2010-09-01
公开日:2021-02-09
发明作者:Arick Brown；Christopher J. Dowd；Asha Nandini Radhamohan
申请人:Genentech, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[0001] This application claims priority for Provisional Patent Application No. 61 / 238,867, filed on September 1, 2009, and for Provisional Patent Application No. 61 / 253,438 filed on October 20, 2009, whose disclosures are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION
[0002] The present invention relates generally to methods for the purification of a polypeptide comprising a CH2 / CH3 region, comprising binding the polypeptide to protein A and eluting with a pH gradient. BACKGROUND OF THE INVENTION
[0003] Large-scale economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using mammalian or bacterial cell lines modified to produce the protein of interest by inserting a recombinant plasmid containing the gene for this protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from animal serum preparations. The separation of the desired protein from the mixture of compounds fed into the cells and the by-products of the cells themselves in a purity sufficient for use as a human therapeutic product represents a formidable challenge.
[0004] The procedures for purifying proteins from cell debris initially depend on the expression site of the protein. Some proteins can be motivated to be secreted directly from the cell into the surrounding growth medium; others are produced intracellularly. For these proteins, the first step in a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such a break releases the entire cell content in the homogenate and, in addition, produces subcellular fragments that are difficult to remove due to their small size. These are usually removed by differential centrifugation or filtration. The same problem arises, albeit to a lesser extent, with proteins directly secreted due to natural cell death and release of intracellular proteins from host cells in the course of the protein production cycle.
[0005] Once a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. Affinity chromatography, which exploits a specific interaction between the protein to be purified and an immobilized capture agent, is normally used for some proteins (for example, proteins for use as a human therapeutic product). Protein A is a useful adsorbent for the affinity chromatography of proteins, such as antibodies, which contain an Fc region. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds with a high affinity (about 10-8 M for human IgG) to the Fc region of antibodies. However, since proteins tend to aggregate or become inflexible, the desired protein (i.e., monomer) is often co-purified with other impurities from these affinity columns, such as protein aggregates, by-products of the cells themselves (ie, the impurities of host cells), or virus filter inlays.
[0006] Other techniques have been developed to further separate these impurities and mixtures of proteins based on their charge, degree of hydrophobicity, or size, such as ion exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography . Several different chromatography resins or sorbents are available for each of these techniques, allowing for the precise adaptation of the purification scheme with the particular protein involved. The essence of each of these separation methods is that proteins can be motivated to move at different rates along a long solid phase (eg, column), which achieves a physical separation that increases when they pass below the solid phase , or selectively adhere to the separation medium, and then differentially eluted by different solvents. However, each of these methods requires additional buffers, resins or sorbents, and other resources for further purification, and these in turn result in longer processing time and higher cost. Thus, more efficient and economical methods for the purification of protein monomers are needed.
[0007] Methods of purifying polypeptides from aggregates, multimers, and modified proteins using a protein A column and eluting with a pH gradient elution system have been described in U.S. Patent Application No. 12 / 008,160.
[0008] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the extent that as if each individual publication, patent and patent application were specifically and individually indicated for so be incorporated by reference. BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides methods for purifying a polypeptide comprising a CH2 / CH3 region by binding the polypeptide to Protein A and eluting with a pH gradient starting at a low pH. These purification methods provide the advantages of achieving better sequential separation of polypeptides or non-aggregates from various impurities, including host cell impurities, virus filter inlays, viruses or virus-like particles, basic polypeptide variants, and aggregates polypeptide, and also greater purity of the desired polypeptide monomers in the purified / pool fraction. These methods can be achieved using various protein A chromatography resins and chromatography sorbents. These methods can also be used on a manufacturing scale and commercial process, and can facilitate the use of downstream purification technologies other than column chromatography.
[00010] In one aspect, the invention provides a method for purifying a polypeptide comprising a CH2 / CH3 region, comprising binding the polypeptide to Protein A and eluting with a pH gradient starting at 5.0 or below.
[00011] In another aspect, the invention provides a method for purifying a polypeptide comprising a CH2 / CH3 region, comprising the steps of: (a) binding the polypeptide to Protein A; and (b) eluting the polypeptide with a pH gradient starting at 5.0 or below using an elution buffer, where the elution buffer contains a high pH buffer and a low pH buffer and where the pH is formed by adjusting a percentage of each pH buffer in the elution buffer.
[00012] In some embodiments, the pH gradient starts around pH 4.2. In other embodiments, the pH gradient starts around pH 4.3. In some embodiments, the pH gradient starts at around 4.6. In some embodiments, the pH gradient ends at or above 3.0. In some embodiments, the pH gradient ends at around 3.7.
[00013] In some embodiments, the high pH buffer is around pH 5.0 and the low pH buffer is around pH 2.7.
[00014] In some embodiments, the percentage of low pH buffer starts at about 35%. In some embodiments, the elution buffer containing the low pH buffer around 35% comprises about 16.25 mM acetate and about 8.75 mM formate. In other embodiments, the percentage of low pH buffer starts at about 25%. In some embodiments, both the elution buffer containing the low pH buffer and around 25% comprises about 18.75 mM acetate and about 6.25 mM formate. In some embodiments, the percentage of low pH buffer starts at about 40%. In some embodiments, the elution buffer containing about 40% low pH buffer comprises about 15 mM acetate and about 10 mM formate.
[00015] In some embodiments, the polypeptide is loaded with a charge density that starts at around 14 g / L. In some embodiments, the polypeptide is loaded with a charge density ranging from about 14 g / L to about 45 g / L.
[00016] In some embodiments, Protein A is a Protein A column chromatography resin or Protein A chromatography sorbent. In some embodiments, Protein A chromatography sorbent is a membrane or monolith .
[00017] In some embodiments, protein A is a Protein A column chromatography resin and wherein the polypeptide has an elution flow rate ranging from about 5 column volumes / hour to about 25 volumes of column / hour.
[00018] In some embodiments, protein A is a Protein A column chromatography resin and wherein a purified fraction of the polypeptide contains about or less than about 12 column volumes of Protein A.
[00019] In some embodiments, a host cell impurity is separated from the polypeptide. In some embodiments, the host cell's impurity is Chinese Hamster Ovarian Protein (CHOP).
[00020] In some embodiments, an aggregate is separated from the polypeptide. In other embodiments, a virus filter inlay is separated from the polypeptide.
[00021] In some embodiments, a virus particle or a virus-like particle is separated from the polypeptide. In other embodiments, a basic polypeptide variant is separated from the polypeptide.
[00022] In some embodiments, the CH2 / CH3 region comprises an immunoglobulin Fc region.
[00023] In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a multi-specific antibody, or an antibody fragment.
[00024] In other embodiments, the polypeptide is an immunoadhesive.
[00025] In some embodiments, the polypeptide has a purity of at least about 98% monomer. In other embodiments, the polypeptide has a purity of at least about 99% monomer.
[00026] In some embodiments, the ratio of a host cell impurity to the purified polypeptide is at least about 75% lower, about 80% lower, about 85% lower, about 90% lower, about 95% lower, about 96% lower, about 97% lower, about 98% lower, or about 99% lower than the ratio in the unpurified polypeptide.
[00027] In some embodiments, a ratio of a host cell impurity to the purified polypeptide is at least about 20% lower than that of a polypeptide purified by a step elution method, wherein the Stepwise elution method comprises binding the polypeptide to Protein A and eluting with a pH starting at 3.6 or below. In some embodiments, a ratio of a host cell impurity to the purified polypeptide is at least about 60% lower than that of a polypeptide purified by a step elution method, wherein the elution method stepwise comprises binding the polypeptide to Protein A and eluting with a pH starting at 3.6 or below.
[00028] In some embodiments, the purified polypeptide has virus particles or virus-like particles that count less than about 15,000 particles / ml. In some embodiments, the purified polypeptide has virus particles or virus-like particles that count less than about 12500 particles / ml, less than about 10,000 particles / ml, less than about 7500 particles / ml, less than about 5000 particles / ml, less than about 2500 particles / ml, less than about 1500 particles / ml, less than about 1000 particles / ml, less than about 750 particles / ml, less than about 500 particles / ml, less than about 250 particles / ml, less than about 100 particles / ml, or less than about 50 particles / ml. In some embodiments, the virus-like particle is a retrovirus-like particle.
[00029] In some embodiments, the purified polypeptide has a viral clearance of a virus or a virus-like particle of at least about 4 LRV (log 10 virus reduction). In some embodiments, the purified polypeptide has viral clearance from a virus or a virus-like particle ranging from about 4 LRV to about 8 LRV. In some embodiments, the purified polypeptide has viral clearance from a virus or a virus-like particle ranging from about 4 LRV to about 7 LRV. In some embodiments, the purified polypeptide has a viral clearance of a virus or a virus-like particle around 5 LRV, around 6 LRV, around 7 LRV, or around 8 LRV. In some embodiments, the virus-like particle is a retrovirus-like particle.
[00030] In some embodiments, the purified polypeptide is a polypeptide monomer.
[00031] In some embodiments, protein A is a modified or unmodified Protein A ligand.
[00032] In some embodiments, purification is a production-scale process.
[00033] In some embodiments of any aspect of the invention, the purification method further comprises subjecting the polypeptide to a virus filtration step or an ion exchange chromatography step. In some embodiments, the ion exchange chromatography step is performed after the purification step.
[00034] In some embodiments of any aspect of the invention, the purification method does not comprise an additional purification step to remove an aggregate.
[00035] In some embodiments of any aspect of the invention, the purification method does not comprise an additional purification step to remove a virus filter fouling.
[00036] In some embodiments of any aspect of the invention, the purification method does not comprise an additional purification step to remove a base polypeptide variant. In some embodiments of any aspect of the invention, the purification method does not comprise an additional purification step to remove an acidic polypeptide variant.
[00037] In another aspect, the invention provides a polypeptide product purified by the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS
[00038] Figure 1 shows the gradient chromatogram of the pH step: the x-axis is in ml of the beginning of the Protein A operation, and the y-axis is the absorbance (mAU). The distinctive shape of the step gradient elution in the 280 UV trace is also shown - large peak is at the beginning of the elution and is then decreased to a stable height and tapers at lower pHs.
[00039] Figure 2 shows the results of SEC (size exclusion chromatography) by the fraction of anti-VEGF antibody # 1. The X axis is the retention time in the SEC column (min), the Y axis is the normalized UV absorbance (mAU). Since the fraction number increased (ie, pH decreases as the gradient elution progresses), HMWS (High Molecular Weight Species) and dimeric peaks (retention times around 12.5 minutes and 13.5 minutes respectively) also increased while the monomeric peak decreased (retention time 16 minutes). The results of these curves were quantified by integrating all the peaks (for example, HMWS, dimer, and monomer), comparing the areas separated from the relative peaks as percentages (for example, the total area was adjusted to 100%, an integration profile of Sample SEC can provide percentages such as "31% HMWS, 36% dimer and 33% monomer").
[00040] Figure 3 shows the result graph of SEC integration for anti-VEGF antibody # 1. This graph shows that the monomer levels were elevated for the first nine fractions of elution with four fractions at 100%, and the levels of dimer and HMWS had peaked later on in the elution. These test results demonstrate that the pH step grandient separates the aggregates from the anti-VEGF # 1 antibody monomer.
[00041] Figure 4A shows the SEC integration profile for the multiple protein molecules (anti-CD20 antibody, anti-VEGF antibody # 2, anti-MUC16 and anti-CD4 antibody). The pH step gradient successfully separated the monomers from the aggregates in the anti-CD20 antibody, anti-VEGF # 2 antibody, anti-MUC16 and anti-CD4 antibody.
[00042] Figure 4B shows the SEC integration profile for a branching anti-Met anti-Met antibody produced by a bacterial host cell (E. coli) fermentation. The pH step gradient successfully separated the aggregated monomers in the anti-aglycosylated Met antibody from a branch.
[00043] Figure 5 shows a side-by-side comparison between a standard step elution of the anti-CD20 antibody (control; elution proteins at pH equal to or less than 3.6 without the pH gradient) and gradient elution of the pH step. The anti-CD20 and CHOP elution graph in the left panel shows CHOP levels per fraction in ppm (parts per million; unit used to standardize the measurement of impurities in quantity of product). The anti-CD20 antibody and aggregate elution graph on the right panel shows the maximum integration values of the SEC per fraction through the gradient elution. The vertical lines on the panels on both the left and right represent the simulation grouping of the contained fractions that should generate a pH gradient elution set with the characteristics shown in the table at the bottom of the slide.
[00044] Figure 6 shows the separation of CHOP for an anti-VEGF antibody # 1. CHOP levels per fraction are expressed in ppm or ng / ml.
[00045] Figure 7 shows the separation of CHOP for an anti-MUC16 antibody. CHOP levels per fraction are expressed in ppm or ng / ml.
[00046] Figure 8 is a layer of chromatogram of Protein A resin MABSELECT ™, MABSELECT SURE ™, PROSEP® Va, PROSEP® Ultra Plus, and POROS® MABCAPTURE ™.
[00047] Figure 9 is a Pareto Plot showing that the% B starting (the starting pH and the gradient of the elution gradient) is the most influential parameter in determining the separation efficiency of aggregates, followed by the density of charge and length of stay.
[00048] Figure 10 is an interaction profile for the parameters of% B starting, charge density and% B starting, total elution length, and residence time.
[00049] Figure 11 is an exemplary manufacturing operation for the gradient elution of the pH step.
[00050] Figure 12 shows the results of the downstream ion exchange membrane study using two cation exchange membrane sizes and three anion exchange membrane sizes. The wedge and nano units are both representative of the fabrication in the number of membrane layers, while the ACRODISC® units have a reduced number of layers, but are the typical bench scale models used.
[00051] Figure 13 shows the comparison of SEC integration results between a 4.1 L column (pilot scale) and a column operation on a 28 ml bench scale.
[00052] Figure 14 is a graph of VIRESOLVE® Pro permeability decline of anti-VEGF antibody # 1 comparing the gradient of the Protein A pH step versus the standard Protein A step in terms of facilitating greater mass production on the VIRESOLVE® Pro parvovirus filter. There was a six-fold increase in mass production over VIRESOLVE® Pro using the protein A pH step gradient.
[00053] Figure 15 is a total pH gradient elution chromatogram of Protein A that shows the actual AKTA UNICORN ™ chromatography software tractions for the complete pH gradient at a charge density of 21 g / L. The initial elevated UV 250 tip at the beginning of the gradient is absent, indicating that the pH at the beginning of the elution is higher than what is necessary to elute the Protein A column products.
[00054] Figure 16 is a pH gradient test AKTA chromatogram of 5.0 to 2.7 (0 to 100% B), which indicates that an anti-VEGF # 1 antibody elutes as a discrete peak in the pH from 4.6 to 3.6.
[00055] Figure 17 is an integration of the peak assay of the ion exchange variant through the gradient elution fractions of the Protein A pH step, which indicates the separation of the basic polypeptide variant at the end of the step elution gradient.
[00056] Figure 18 is the RVLP (Particle as Retrovirus) particle count from the QPCR (Quantitative Polymerase Chain Reaction) analysis by fraction plotted against the elution of the anti-VEGF # 1 antibody product. Most RVLPs have eluted recently on the gradient where little product elution has occurred.
[00057] Figure 19 is LRV (log 10 virus reduction) for each fraction in the gradient elution of the anti-VEGF # 1 Protein A pH step.
[00058] Figure 20 shows the cumulative LRVs for simulated groups of the fraction, showing that the largest LRVs can be achieved in the Protein A set if the subsequent fractions are omitted. DETAILED DESCRIPTION OF THE INVENTION
[00059] The present invention provides methods for purifying a polypeptide comprising a CH2 / CH3 region by binding the polypeptide to Protein A and eluting with a pH gradient starting at a low pH. The inventors made the surprising discovery that elution polypeptides comprising a Protein A CH2 / CH3 region with a pH gradient at a low pH can provide better sequential separation of polypeptides from various impurities, including host cell impurities , virus, virus or virus-like particle filter inlays, basic polypeptide variants, and / or polypeptide aggregates, and also achieve greater purity or percentage of the desired polypeptide monomers in the purified / pool fraction. Thus, the invention has significant advantages. The inventors also found that these methods can be achieved using various Protein A chromatography resins and chromatography sorbents and that these methods can be used in a scale of manufacture and commercial process, and can facilitate the use of different alternative downstream purification technologies column chromatography (eg membrane adsorbents).
[00060] Accordingly, in one aspect of the invention, a method is provided for purifying a polypeptide comprising a CH2 / CH3 region, which comprises binding the polypeptide to Protein A and eluting with a pH gradient starting at 5.0 or below.
[00061] In another aspect of the invention, a method is provided for the purification of a polypeptide comprising a CH2 / CH3 region, comprising the steps of: (a) binding the polypeptide to Protein A, and (b) eluting the polypeptide with a pH gradient starting at 5.0 or below using an elution buffer, where the elution buffer contains a high pH buffer and a low pH buffer and where the pH gradient is formed by adjusting a percentage of each pH buffer in the elution buffer.
[00062] In yet another aspect of the invention, a polypeptide purified by the methods described herein is provided.
[00063] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., Eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., Eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., Eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A.Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., Ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995). Definitions
[00064] It is understood that the polypeptide of interest here is one that comprises a CH2 / CH3 region and, therefore, is capable of purification by Protein A. The term "CH2 / CH3 region" when used here refers to those residues of amino acid in the Fc region of an immunoglobulin molecule that interacts with Protein A. In some embodiments, the CH2 / CH3 region comprises an intact CH2 region followed by an intact CH3 region, and more preferably comprises an immunoglobulin Fc region. Examples of proteins containing the CH2 / CH3 region include antibodies, immunoadhesins and fusion proteins comprising a protein of interest fused or conjugated to a CH2 / CH3 region.
[00065] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be disrupted by non-amino acids. The terms also cover an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
[00066] As used herein, the term "purified polypeptide" or "purified protein" is a product eluted from Protein A affinity chromatography using the pH gradient methods as described herein. The purified polypeptides / proteins preferably contain mainly polypeptide monomers.
[00067] As used herein, the term "unpurified polypeptide", "unpurified protein", or "protein filler" is a polypeptide or protein in the loading material or starting material prior to the purification step by the affinity chromatography of the Protein A.
[00068] As used herein, the term "impurity" or "impurities" is a material that is different from the desired polypeptide monomer product. Impurities include, but are not limited to, a polypeptide variant (e.g., acidic or basic polypeptide variant), polypeptide fragment, aggregate or derivative of the desired polypeptide monomer, other polypeptide, lipid, nucleic acid, endotoxin, impurity of the host cell, or inlaying the virus filter.
[00069] As used herein, the term "monomer" refers to a single unit of a polypeptide comprising a CH2 / CH3 region. For example, in the case of an antibody, a monomer consists of two heavy chains and two light chains; in the case of an unbranched antibody, a monomer consists of a heavy chain and a light chain.
[00070] As used herein, the term "basic polypeptide variant" or "basic variant" refers to a variant of a polypeptide of interest that is more basic (for example, as determined by cation exchange chromatography) than polypeptide of interest.
[00071] As used herein, the term "acidic polypeptide variant" or "acidic variant" refers to a variant of a polypeptide of interest that is more acidic (for example, as determined by cation exchange chromatography) than polypeptide of interest.
[00072] As used herein, the term "aggregate" refers to any multimers of a polypeptide or a polypeptide fragment comprising a CH2 / CH3 region. For example, an aggregate can be a dimer, trimer, tetramer, or a multimer larger than a tetramer, etc.
[00073] As used herein, the term "host cell impurity" refers to any proteinaceous contaminant or by-product introduced by the host cell line, cultured cell fluid, or cell culture. Examples include, but are not limited to, Chinese Hamster Ovary Protein (CHOP), E. coli Protein, yeast protein, simian COS protein, or myeloma cell protein (for example, NS0 protein ( mouse plastocytoma cells derived from a BALB / c mouse)).
[00074] As used herein, the term "virus filter inlay" refers to any large molecular weight particle or high molecular weight species (HMWS) with a hydrodynamic diameter similar to or greater than the pore size distribution of the parvovirus filter. Virus filter inlays include, but are not limited to, soluble aggregates of high molecular weight polypeptide and soluble and / or insoluble aggregates of host cell impurities (e.g., CHOP).
[00075] The "host cell" includes an individual cell or cell culture that can be or was a recipient for the vector for incorporating polynucleotide inserts to produce polypeptides. Host cells include the offspring of a single host cell, and the offspring may not necessarily be completely identical (in morphology or in the complement of genomic DNA) to the original source cell due to natural, accidental, or deliberate mutation.
[00076] The "solid phase", as used herein, refers to a non-aqueous matrix to which Protein A can adhere.
[00077] A "buffer" is a buffered solution that resists changes in pH by the action of its conjugated acid-base components. Various buffers that can be employed depending, for example, on the desired pH of the buffer, are described in A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., Ed. Calbiochem Corporation (1975).
[00078] The "equilibrium buffer" here is that used to prepare the solid phase (with immobilized Protein A) to load the protein of interest.
[00079] The "wash buffer" is used here to refer to the buffer that is passed over the solid phase (with immobilized Protein A) following loading and before the elution of the protein of interest.
[00080] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, provided that they retain, or are modified to understand, a CH2 / CH3 region as defined herein.
[00081] The "antibody fragments" comprise a part of a full length antibody, in general its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; single chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments. As used herein, the antibody fragment comprises a CH2 / CH3 region.
[00082] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except with respect to possible naturally occurring mutations that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the present invention can be produced by the hybridoma method first described by Kohleret al, Nature 256: 495 (1975), or they can be produced by recombinant DNA methods (see, for example , US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example.
[00083] Monoclonal antibodies here specifically include "chimeric" antibodies (immunoglobulins) in which a part of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of particular antibody, while the rest of the chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (US Patent no. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
[00084] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 - 35 (1H), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a "hypervariable circuit" (ie residues 26-32 (L1), 5052 (L2) and 91-96 (L3) in the chain variable domain light and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The "structure" or "FR" residues are those residues of variable domain different from the residues of the hypervariable region as defined herein.
[00085] "Humanized" forms of non-human antibodies (eg, murine) are chimeric antibodies that contain the minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-primate. human being having the desired specificity, affinity and capacity. In some cases, residues of the human immunoglobulin Fv (FR) structure region are replaced by the corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not seen in the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable circuits correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a sequence of human immunoglobulin. The humanized antibody optionally will also comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
[00086] As used herein, the term "immunoadhesin" means antibody-like molecules that combine the "binding domain" of a heterologous "adhesin" protein (for example, a receptor, ligand, or enzyme) with the effector functions of a immunoglobulin constant domain. Structurally, immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is otherwise the recognition of the antigen and binding site (antigen combining site) of an antibody (i.e., it is "heterologous" ) and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin is preferably derived from the heavy chains y1, Y2, or Y4 since the immunoadhesins comprising these regions can be purified by Protein A chromatography (Lindmark et al., J. Immunol. Meth. 62 : 1-13 (1983)).
[00087] The term "ligand-binding domain" as used herein refers to any native cell surface recipient or any region or derivative thereof that retains at least one qualitative ligand linkage from a corresponding native recipient. In a specific embodiment, the recipient is a cell surface polypeptide having an extracellular domain that is homologous to a member of the immunoglobulin supergenefamily. Other recipients, who are not members of the immunoglobulin supergenefamily, but are nevertheless specifically covered by this definition, are recipients for cytokines, and in particular recipients with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and superfamily families growth factor receptor, and cell adhesion molecules, for example, (E-, L- and P-) selectins.
[00088] The term "receptor binding domain" is used to designate any native ligand for a receptor, including cell adhesion molecules, or any region or derivative of such native ligand that retains at least one receptor binding capacity qualitative nature of a corresponding native ligand. This definition, among others, specifically includes the ligand-binding sequences for the aforementioned receptors.
[00089] An "antibody immunoadhesin chimera" comprises a molecule that combines at least one antibody binding domain (as defined herein) with at least one immunoadhesin (as defined in this application). Exemplary antibody immunoadhesin chimeras are the bispecific CD4-IgG chimeras described in Berg et al., PNAS (USA) 88: 4723-4727 (1991) and Chamow et al., J. Immunol. 153: 4268 (1994).
[00090] For use here, unless expressly stated otherwise, the use of the terms "one", "one", and so on refers to one or more.
[00091] Reference to "about" a value or parameter here includes (and describes) the embodiments that are directed to that value or parameter per se. For example, the description that refers to "about X" includes the description of "X". The numerical ranges are inclusive of the numbers that define the range.
[00092] It is understood that wherever the embodiments are described herein with the language "comprising", the different analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Polypeptide Purification
[00093] The process here involves purifying a polypeptide containing the CH2 / CH3 region of one or more impurities by Protein A affinity chromatography using a pH gradient that starts from a low pH. In one aspect, the polypeptide comprising a CH2 / CH3 region can be purified by a method that comprises binding the polypeptide to Protein A and eluting with a pH gradient starting at 5.0 or below.
[00094] In another aspect, the polypeptide comprising a CH2 / CH3 region can also be purified by a method comprising the steps of: (a) binding the polypeptide to Protein A; and (b) eluting the polypeptide with a pH gradient starting at 5.0 or below using an elution buffer, where the elution buffer contains a high pH buffer and a low pH buffer and where the pH is formed by adjusting a percentage of each pH buffer in the elution buffer.
[00095] Protein A can be a modified or unmodified Protein A ligand. As used herein, "Protein A ligand" encompasses native Protein A, Protein A produced synthetically (for example, by peptide synthesis or by recombinant techniques), and variants that retain the ability to bind to proteins that have a region CH2 / CH3. A modified Protein A ligand can be chemically designed to be stable in high pH solutions for small amounts of time (eg, MABSELECT SURE ™ (GE Healthcare (Piscataway, NJ)), POROS® M AB CAPTURE ™ A (Applied Biosystems (Foster City, CA)) The term "unmodified Protein A ligand", as used herein, encompasses Protein A ligand that is similar to Protein A recovered from a native source. For example, MABSELECT ™, PROSEP ™ Va, PROSEP ™ Ultra Plus, can be purchased commercially from GE Healthcare (Piscataway, NJ) or Millipore (Billerica, MA).
[00096] Protein A can be immobilized in a solid phase. The solid phase can be a purification column, a discontinuous phase or discrete particles, a membrane, or filter. Examples of materials for the formation of the solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (for example, controlled porous glass), poly (styrenedivinol) benzene, polyacrylamide, ceramic particles, and derivatives of any of the above.
[00097] Protein A immobilized on a solid phase is used to purify polypeptides containing the CH2 / CH3 region. In some embodiments, the solid phase is a Protein A column resin comprising a Protein A based resin and globules, silica based resin or agarose based resin to immobilize Protein A. For example, the phase solid is a column of controlled porous glass or a column of silicic acid. Sometimes, the column was coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence to the column. The PROSEP ™ A column is an example of a Protein A controlled porous glass column that is coated with glycerol. In other embodiments, the solid phase is a Protein A chromatography sorbent to immobilize Protein A. Protein A chromatography sorbents include, but are not limited to, membranes (e.g., Sartorius (Goettingen, Germany ), Protein A SARTOBIND ™ membrane) or monoliths (eg, BIA Separations (Villach, Austria), HLD Protein A CIM® monoliths).
[00098] The solid phase for Protein A chromatography can be equilibrated with an equilibration buffer, and unpurified polypeptides comprising various impurities (for example, harvested cell culture fluid) can then be loaded into the balanced solid phase. The polypeptide can be loaded with a loading buffer. Conveniently, the balance buffer to balance the solid phase can be the same as the charge buffer, but this is not necessary. When polypeptides circulate through the solid phase, polypeptides and various impurities are adsorbed on immobilized protein A. Wash buffers can be used to remove some impurities, such as host cell impurities, but not the polypeptides of interest.
[00099] The balance buffer is preferably isotonic and commonly has a pH in the range of about 6 to about 8. For example, a balance buffer can have 25 mM Tris, 25 mM NaCl, 5 mM EDTA, and pH 7.1.
[000100] The "loading buffer" refers to a buffer that is used to load the protein mixture containing the CH2 / CH3 region and contaminants in the solid phase in which the protein is immobilized. Often, the balance and charge plugs are the same.
[000101] The wash buffer can serve to elute the impurity of the cell line or other various impurities. The conductivity and / or pH of the wash buffer is such that impurities are eluted from Protein A chromatography, but not any significant amount of the polypeptide of interest.
[000102] Protein A-bound polypeptide can be eluted with a pH gradient using a single elution buffer or a combination of elution buffers.
[000103] The "elution buffer" is used to elute the polypeptide containing the immobilized Protein A CH2 / CH3 region. As used herein, the elution buffer contains a high pH buffer and a low pH buffer and thus forms a pH gradient by adjusting a percentage of the high pH buffer and the low pH buffer in the elution buffer. In some embodiments, the elution buffer has a pH in the range of about 3 to about 5. The pH values as used herein are measured without the presence of polypeptides. Examples of pH buffers that control pH within this range include, but are not limited to, phosphate, acetate, citrate, formic acid and ammonium buffers, as well as combinations thereof. Such preferred buffers are acetate and formic acid buffers.
[000104] In some embodiments, the pH gradient starts at around 5.0. In some embodiments, the pH gradient starts below 5.0. In some embodiments, the pH gradient starts varying from about 5.0 to about 4.0. In some embodiments, the pH gradient starts at about 4.9, about 4.8, about 4.7, about 4.6, about 4.5, about 4.4, about 4.3, about 4.2, about 4.1, or about 4.0. In some embodiments, the pH gradient starts at about 4.98, about 4.96, about 4.94, about 4.92, about 4.90, about 4.88, about 4.86, about 4.84, about 4.82, about 4.80, about 4.78, about 4.76, about 4.74, about 4.72, about 4, 70, about 4.68, about 4.66, about 4.64, about 4.62, about 4.60, about 4.58, about 4.56, about 4.54, about 4.52, about 4.50, about 4.48, about 4.46, about 4.44, about 4.42, about 4.40, about 4.40, about 4.38, about 4.36, about 4.34, about 4.32, about 4.30, about 4.28, about 4.24, about 4.22, about 4.22, about 4.20, about 4, 18, about 4.16, about 4.14, about 4.12, about 4.10, about 4.08, about 4.06, about 4.04, or about 4.02 .
[000105] In some embodiments, the pH gradient ends at around 3.0. In some embodiments, the pH gradient ends above 3.0. In some embodiments, the pH gradient ends up varying from about 3.0 to about 4.0. In some embodiments, the pH gradient ends at about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9. In some embodiments, the pH gradient ends at about 3.12, about 3.14, about 3.16, about 3.18, about 3.20, about 3.22, about 3.22, about 3.24, about 3.26, about 3.28, about 3.30, about 3.32, about 3.34, about 3.36, about 3.38, about 3.38, about 3, 40, about 3.42, about 3.44, about 3.46, about 3.48, about 3.50, about 3.52, about 3.54, about 3.56, about 3.58, about 3.60, about 3.61, about 3.62, about 3.63, about 3.64, about 3.65, about 3.66, about 3.67, about 3.68, about 3.69, about 3.70, about 3.71, about 3.72, about 3.73, about 3.74, about 3. 75, about 3.76, about 3.77, about 3.78, about 3.79, about 3.80, about 3.82, about 3.84, about 3.84, about 3.86, about 3.88, about 3.9, about 3.92, about 3.94, about 3.96, or about 3.98.
[000106] In some embodiments, the pH gradient starts around pH 4.2 and ends around pH 3.7. In some embodiments, the pH gradient starts around 4.24 and ends around pH 3.69. For example, anti-VEGF antibodies, anti-CD20 antibodies, anti-MUC16 antibodies, anti-CD4 antibodies, and anti-Met antibodies from a branch can be purified using the pH gradient starting around pH 4.24 and ends around pH 3.69.
[000107] In other embodiments, the pH gradient starts around pH 4.3 and ends around pH 3.7. In some embodiments, the pH gradient (i.e., pH step gradient) starts around pH 4.34 and ends around pH 3.69. For example, anti-VEGF antibodies, anti-CD20 antibodies, anti-MUC16 antibodies, anti-CD4 antibodies, and anti-Met antibodies from a branch can be purified using the pH gradient that starts at about pH 4.34 and ends at about pH 3.69.
[000108] In some embodiments, the pH gradient starts around pH 4.6 and ends around pH 3.7. In some embodiments, the pH gradient (i.e., total pH gradient) starts around pH 4.58 and ends around pH 3.69. For example, anti-VEGF antibodies, anti-CD20 antibodies, anti-MUC16 antibodies, anti-CD4 antibodies, and anti-Met antibodies from a branch can be purified using the pH gradient starting around pH 4.58 and ends around pH 3.69.
[000109] The elution buffer contains a high pH buffer and a low pH buffer and the pH gradient is formed by adjusting a percentage of each pH buffer in the elution buffer. In some embodiments, the high pH buffer is around 5.0 and the low pH buffer is around 2.7. For example, the high pH buffer can be 25 mM acetate and pH 5.0 and the low pH buffer can be 25 mM formic acid and pH 2.7.
[000110] Adjusting the starting percentage of the low pH buffer can optimize and maximize the purity of the purified polypeptide, as well as the sequential separation of impurities, including aggregates, cell line impurities, basic polypeptide variant, virus particles, similar particles to viruses, and virus filter inlays, of the polypeptide monomers. The percentage of low pH buffer in the elution buffer can start at about 25%, about 30%, about 35%, about 40%, or about 45%.
[000111] In some embodiments, the percentage of low pH buffer in the elution buffer can start at around 25%. In some embodiments, the elution buffer containing the low pH buffer around 25% comprises about 19 mM acetate, about 6 mM formate, and buffer conductivity around 1140 at pH 4.5 to 4, 6. For example, the elution buffer containing the low pH buffer around 25% comprises 18.75 mM acetate, 6.25 mM formate, buffer conductivity of 1141 uS / cm, at pH 4.58.
[000112] In some embodiments, the percentage of low pH buffer in the elution buffer can start at around 35%. In some embodiments, the elution buffer containing the low pH buffer around 35% comprises about 16 mM acetate, about 9 mM formate, and buffer conductivity around 1040 at pH 4.3 to 4, 4. For example, the elution buffer containing the low pH buffer around 35% comprises 16.25 mM acetate, 8.75 mM formate, buffer conductivity of 1039 uS / cm, at pH 4.34.
[000113] In some embodiments, the percentage of low pH buffer in the elution buffer can start around 40%. In some embodiments, the elution buffer containing about 40% low pH buffer comprises about 15 mM acetate, about 10 mM formate, and buffer conductivity at about 974 at pH 4.2 to 4.3 . For example, the elution buffer containing the low pH buffer around 40% comprises 15 mM acetate, 10 mM formate, buffer conductivity of 974 uS / cm, at pH 4.24.
[000114] In some embodiments, the percentage of low pH buffer in the elution buffer can end up to around 60%. In some embodiments, the elution buffer containing the low pH buffer around 60% comprises about 10 mM acetate, about 15 mM formate, and buffer conductivity at about 763 at pH 3.6 to 3.7 . For example, the low pH buffer at the end of the pH gradient can be 10 mM acetate, 15 mM formate, buffer conductivity of 763 uS / cm, at pH 3.69.
[000115] In some embodiments, the elution buffer has a buffer conductivity ranging from about 1200 uS / cm to about 500 uS / cm. In some embodiments, the elution buffer has a buffer conductivity ranging from about 1150 uS / cm to about 700 uS / cm. In some embodiments, the elution buffer has a buffer conductivity of about 1145 uS / cm, about 1141 uS / cm, about 1130 uS / cm, about 1120 uS / cm, about 1110 uS / cm , about 1000 uS / cm, about 1039 uS / cm, about 1000 uS / cm, about 974 uS / cm, about 900 uS / cm, about 800 uS / cm, about 763 uS / cm, or about 700 uS / cm.
[000116] In some embodiments, the composition of the elution buffer is about 9 to 20 mM acetate and 5 to 15 mM formate. In some embodiments, the elution composition is about 10 to 19 mM acetate and 6 to 16 mM formate.
[000117] Adjusting the charge density of the polypeptide can also optimize and maximize the purity of the purified polypeptide and the separation of impurities, including aggregates, cell line impurities, basic polypeptide variant, virus particles, virus-like particles, and virus filter inlays, of the polypeptide monomers.
[000118] The term "charge density" or "charge density" is the density of the purified polypeptide (g) per liter of chromatography resin or the density of the purified polypeptide per liter of membrane / filter volume (L). The charge density is measured in g / L.
[000119] In some embodiments, the polypeptide is loaded with a charge density that starts at 14 g / L or above. In some embodiments, the polypeptide is loaded with a charge density ranging from about 14 g / L to about 45 g / L or from about 14 g / L to about 70 g / L. In some embodiments, the polypeptide is loaded with a charge density of about 15 g / L, about 17 g / L, about 19 g / L, about 21 g / L, about 23 g / L , about 25 g / l, about 26 g / l, about 27 g / l, about 28 g / l, about 29 g / l, about 31 g / l, about 33 g / l, about 35 g / l, about 37 g / l, about 39 g / l, about 41 g / l, about 43 g / l, about 45 g / l, about 50 g / l, about 55 g / L, about 60 g / L, about 65 g / L, or about 70 g / L.
[000120] Adjusting the polypeptide elution residence time (or the elution flow rate) can also optimize and maximize the polypeptide purity and the sequence separation of impurities from the polypeptide monomers. In the increased charge density, the polypeptide elution residence time plays a much greater role in the pH gradient's ability to fractionate the aggregates efficiently. In some embodiments, the polypeptide has an elution flow rate ranging from about 5 column volumes / hour to about 35 column volumes / hour. In some embodiments, the polypeptide has an elution flow rate ranging from about 5 column volumes / hour to about 25 column volumes / hour. In some embodiments, the polypeptide has an elution flow rate of about 5 column volumes / hour, about 7.5 column volumes / hour, about 10 column volumes / hour, about 12.5 column volumes / hour, about 15 column volumes / hour, about 17.5 column volumes / hour, about 20 column volumes / hour, about 22.5 column volumes / hour, about 25 volumes column / hour, about 27.5 column volumes / hour, about 30 column volumes / hour, about 32.5 column volumes / hour, or about 35 column volumes / hour.
[000121] Purified polypeptides using the methods described herein have a yield of at least about any 75% non-purified polypeptide, 80% non-purified polypeptide, 85% non-purified polypeptide, 90% non-purified polypeptide , 95% of unpurified polypeptide, 96% of unpurified polypeptide, 97% of unpurified polypeptide, 98% of unpurified polypeptide, or 99% of unpurified polypeptide.
[000122] Yield is the total amount of purified polypeptide collected compared to the unpurified polypeptide prior to purification from Protein A affinity chromatography as described herein, generally expressed as a percentage of the unpurified polypeptide.
[000123] In some embodiments, the ratio of a host cell impurity to the purified polypeptide is at least about 75% lower, about 80% lower, about 85% lower, about 90% more low, about 95% lower, about 96% lower, about 97% lower, about 98% lower, or about 99% lower than the ratio in the unpurified polypeptide.
[000124] In some embodiments, the ratio of a host cell impurity to the purified polypeptide is at least about 20% lower, about 30% lower, about 40% lower, about 50% more low, about 60% lower, or about 70% lower than the ratio in the purified polypeptide using a pH purification step different from that of the present invention. For example, in a conventional or typical Protein A elution method, the polypeptide is purified by binding the polypeptide to Protein A and eluting the polypeptide at pH 3.6 or below without the pH gradient. Consequently, in some embodiments, the ratio of a host cell impurity to the purified polypeptide is at least about 20% lower, about 30% lower, about 40% lower, about 50% lower , about 60% lower, or about 70% lower than the ratio in a polypeptide purified by a step elution method, wherein the step elution method comprises binding the polypeptide to Protein A and eluting with a pH that starts at 3.6 or below.
[000125] In some embodiments, the ratio of a virus filter inlay to the purified polypeptide is at least about 75% lower, at least about 80% lower, about 85% lower, about 90% lower, about 95% lower, about 96% lower, about 97% lower, about 98% lower, or about 99% lower than the ratio in the unpurified polypeptide.
[000126] In some embodiments, the ratio of a virus filter inlay to the purified polypeptide is at least about 20% lower, about 30% lower, about 40% lower, about 50 % lower, about 60% lower, or about 70% lower than the ratio in the purified polypeptide using a pH purification step different from the present invention. For example, in a conventional or typical step A protein elution method, the polypeptide is purified by binding the polypeptide to Protein A and eluting the polypeptide at pH 3.6 or below without the pH gradient. Consequently, in some embodiments, the ratio of a virus filter inlay to the purified polypeptide is at least about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower than the ratio in a polypeptide purified by a step elution method, wherein the step elution method comprises binding the polypeptide to Protein A and elution with a pH starting at 3.6 or below.
[000127] In some embodiments, the purified polypeptide has a virus particle or virus-like particle that counts less than about 15000 particles / ml. In some embodiments, the purified polypeptide has a virus particle or virus-like particle that counts less than about 12500 particles / ml, less than about 10,000 particles / ml, less than about 7500 particles / ml, less than about 5000 particles / ml, less than about 2500 particles / ml, less than about 1500 particles / ml, less than about 1000 particles / ml, less than about 750 particles / ml, less than about 500 particles / ml, less than about 250 particles / ml, less than about 100 particles / ml, or less than about 50 particles / ml. In some embodiments, the virus-like particle is a retrovirus-like particle.
[000128] As used herein, the term "virus particle" is a virion consisting of a nucleic acid nucleus surrounded by a protective layer of protein (capsid). "Virus-like particles" are non-infectious viruses that resemble morphological, biochemical or other similar properties. They are defective in at least one of the components needed for the virus life cycle. An example of a virus-like particle is a retrovirus-like particle that cannot replicate. The virus particle or virus-like particle can be endogenous or exogenous (casual). A virus particle or endogenous virus-like particle is produced by a host cell line, present in cells and cell culture fluid, and can be considered as a host cell impurity. An exogenous or casual virus or virus-like particle is not derived from a host cell line.
[000129] In some embodiments, the purified polypeptide has viral clearance of a virus or a virus-like particle of at least about 4 LRV (log 10 virus reduction). In some embodiments, the purified polypeptide has viral clearance from a virus or a virus-like particle ranging from about 4 LRV to about 8 LRV. In some embodiments, the purified polypeptide has viral clearance from a virus or a virus-like particle ranging from about 4 LRV to about 7 LRV. In some embodiments, the purified polypeptide has a viral clearance of a virus or a virus-like particle around 5 LRV, around 6 LRV, around 7 LRV, or around 8 LRV. In some embodiments, the virus-like particle is a retrovirus-like particle.
[000130] As used herein, LRV is the difference of log 10 (total virus) in the unpurified polypeptide and in the purified polypeptide.
[000131] In some embodiments, the purified polypeptide is a polypeptide monomer.
[000132] In some embodiments, a purified fraction of the polypeptide contains around or less than about 20 column volumes of Protein A. In some embodiments, a purified fraction of the polypeptide contains around or less than about 15 column volumes of Protein A. In some embodiments, a purified fraction of the polypeptide contains around or less than about 12 column volumes of Protein A. In some embodiments, a purified fraction of the polypeptide contains at least around or less than about 11, about 10, about 9, about 8, about 7, about 6, about 5.5, or about 5.0 column volumes of Protein A.
[000133] In some embodiments, the methods described herein remove at least two of the impurities described herein from the desired polypeptide monomer product. For example, the methods remove both an aggregate and an impurity from the host cell line, both an aggregate and a virus filter inlay, both an aggregate and a virus particle, both an aggregate and a virus-like particle, both a aggregated as a variant of basic polypeptide, or an impurity of the host cell line and a virus particle, etc. In some embodiments, the methods described herein remove at least three of the impurities described herein from the desired polypeptide monomer product. For example, the methods remove an aggregate, a host cell impurity, and a virus filter inlay, or an aggregate, a host cell impurity, and a virus particle, and a basic polypeptide variant, etc. In some embodiments, the methods described herein remove at least four of the impurities described herein from the desired polypeptide monomer product. For example, the methods remove an aggregate, a host cell impurity, a virus filter inlay, and a virus particle, or an aggregate, a host cell impurity, a virus filter inlay, and a particle similar to virus. In some embodiments, the methods described herein remove at least five of the impurities described herein from the desired polypeptide monomer product. For example, the methods remove an aggregate, a host cell impurity, a virus filter inlay, a virus particle, and a virus-like particle, etc. In some embodiments, the methods described herein remove all impurities from the desired polypeptide product.
[000134] In some embodiments, the methods described herein do not comprise an additional purification step to remove an aggregate, and the purified polypeptides have a purity of at least about 98% or about 99% monomer. Purification of the aggregate normally performed in a separate ion exchange chromatography step is not required following protein A chromatography using the pH gradient as described above.
[000135] In some embodiments, the methods described herein do not comprise an additional purification step to remove a virus filter encrustation, and the purified polypeptides have a purity of at least about 98% or about 99% monomer .
[000136] In some embodiments, the purification method does not comprise an additional purification step to remove a basic or acidic polypeptide variant.
[000137] The polypeptide purified using the methods described herein can be subjected to additional purification steps before, during or after the Protein A chromatography step. Other exemplary purification steps include, but are not limited to, hydroxylapatite chromatography; dialysis; affinity chromatography using an antibody to capture the protein; hydrophobic interaction chromatography (HIC) (for example, fractionation in an HIC); precipitation with ammonium sulfate; precipitation of polyethylene glycol or derivative of polyethylene glycol, anionic or cationic exchange chromatography; ethanol precipitation; Reverse phase HPLC; chromatography on silica; chromatofocusing; SDS-PAGE chromatography, virus filtration, gel filtration, and weak division.
[000138] In some embodiments, the polypeptides are further subjected to a virus filtration step. For example, a parvovirus filter can be used in the virus filtration step after the Protein A chromatography step using a pH gradient as described herein.
[000139] In some embodiments, the polypeptides are further subjected to an ion exchange chromatography step. In some embodiments, the ion exchange chromatography step comprises a cation exchange chromatography step. In some embodiments, the ion exchange chromatography step comprises an anion exchange chromatography step. In some embodiments, the ion exchange chromatography step comprises a cation exchange chromatography step and an anion exchange chromatography step.
[000140] In some embodiments, the ion exchange chromatography step operates continuously after the Protein A chromatography step as described herein. For example, the cation and anion exchange chromatography membranes can be used instead of the standard cation and / or anion exchange chromatography columns following the Protein A chromatography method described here to achieve purified polypeptides of comparable purity and yield produced by Standard Protein A chromatography method without the pH gradient followed by the standard cation and anion exchange column chromatography steps.
[000141] In some embodiments, the methods described here are manufacturing or commercial processes and scale. As used herein, manufacturing-scale or commercial processes refer to a large-scale purification of protein / polypeptide, for example, in a protein / polypeptide product on a fermentation scale of about 1 kL to about 25 kL per process of purification. Polypeptides
[000142] The polypeptide or protein to be purified using the methods described herein includes, but is not limited to, antibody, immunoadhesin, or a polypeptide fused or conjugated to a CH2 / CH3 region. Techniques for generating such molecules are discussed below. Antibodies.
[000143] Antibodies within the scope of the present invention include, but are not limited to: anti-CD20 antibodies such as chimeric anti-CD20 "C2B8" as in US Patent No. 5,736,137 (RITUXAN); anti-VEGF antibodies, including mature humanized and / or affinity anti-VEGF antibodies such as the huA4.6.1 AVASTIN® humanized anti-VEGF antibody (Kim et al., Growth Factors, 7: 53-64 (1992), Publication International No. WO 96/30046, and WO 98/45331, published Oct. 15, 1998) and V3LA; anti-MUC16 antibody; anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum. 39 (1): 52-56 (1996)) and the Ibalizumab antibody (TNX355); anti-MET antibodies such as the branch 5D5 anti-C-Met antibody; anti-HER2 Trastuzumab antibodies (HERCEPTIN®) (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 42854289 (1992), US Patent No. 5,725,856) and humanized 2C4 (WO 01/00245, Adams et al.), a chimeric or humanized variant of the 2H7 antibody as in US Patent No. 5,721,108 B1, or Tositumomab (BEXXAR®); anti-IL-8 antibodies (St John et al., Chest, 103: 932 (1993), and International Publication No. WO 95/23865); anti-prostate stem cell (PSCA) antigen antibodies (WO 01/40309); anti-CD40 antibodies, including S2C6 and their humanized variants (WO 00/75348); anti-CDl antibodies (US Patent No. 5,622,700, WO 98/23761, Steppe et al., Transplant Intl. 4: 3-7 (1991), and Hourmant et al., Transplantation 58: 377-380 (1994)) ; anti-CD18 (US Patent No. 5,622,700, published April 22, 1997, or as in WO 97/26912, published July 31, 1997); anti-IgE antibodies (including E25, E26 and E27; US Patent No. 5,714,338, published on Feb. 3, 1998 or US Patent No. 5,091,313, published on Feb. 25, 1992, WO 93/04173 published on 04 Mar. 1993, or International Application No. PCT / US98 / 13410 filed June 30, 1998, US Patent No. 5,714,338, Presta et al., J. Immunol. 151: 2623-2632 (1993), and International Publication No. WO 95/19181); anti-Apo-2 receptor antibodies (WO 98/51793 published 18 Nov. 1998); anti-TNF- antibodies, including cA2 (REMICADE®), CDP571 and MAK-195 (See, US Patent No. 5,672,347 published September 30, 1997, Lorenz et al. J. Immunol. 156 (4): 1646-1653 (1996), and Dhainaut et al., Crit Care Care 23 (9): 1461-1469 (1995)); anti-tissue factor (TF) antibodies (European Patent No. 0 420 937 B l issued on Nov. 9, 1994); anti-human integrin antibodies a4B7 (WO 98/06248 published Feb. 19, 1998); anti-epidermal growth suit (EGFR) antibodies (for example, chimerized or humanized antibody 225 as in WO 96/40210 published on Dec. 19, 1996); anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893 published May 7, 1985); anti-CD25 or anti-Tac antibodies such as CHI-621 (SIMULECT® and ZENAPAX® (See US Patent No. 5,693,762 published Dec 2, 1997); anti-CD52 antibodies such as CAMPATH-IH (Riechmann et al Nature 332: 323-337 (1988)); anti-Fc receptor antibodies such as the M22 antibody directed against Fey RI as in Graziano et al. J. Immunol. 155 (10): 4996-5002 (1995); of anti-carcinoembryonic antigen (CEA) such as hMN-1 4 (Sharkey et al. Cancer Res. 55 (23Suppl): 5935s-5945s (1995); antibodies directed against breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al. Cancer Res. 55 (23): 5852s-5856s (1995); and Richman et al. Cancer Res. 55 (23 Supp): 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al. Eur J Immunol. 26 (1): 1-9 (1996)); anti-CD38 antibodies, for example, AT 13/5 (Ellis et al. J. Immunol 155 (2): 925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55 ( 23 Suppl): 5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al. Cancer Res 55 (23 Suppl): 5899s-5907s (1995)); anti-EpCAM antibodies such as 17-1A (PANOREX®); anti-GpIIb / IIIa antibodies such as abciximab or c7E3 Fab (REOPRO); anti-RSV antibodies such as MEDI-493 (SYNAGIS®); anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such as PR0542; anti-hepatitis antibodies such as the anti-Hep B OSTAVIR® antibody; anti-CA 125 antibodies, such as OvaRex; anti-idiotypic GD3 BEC2 epitope antibody; anti-avB3 antibodies, including VITAXIN; anti-human renal cell carcinoma antibody such as ch-G250; ING-1; anti-human 17-1 A antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human R24 melanoma antibody directed against ganglioside GD3; anti-human squamous cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibodies such as Smart ID 10 and the anti-HLA antibody DR Oncolym (Lym-1).
[000144] With the exception of the antibodies specifically identified above, the skilled practitioner can generate antibodies directed against an antigen of interest, for example, using the techniques described below. (i) Antigen Selection and Preparation
[000145] The antibody contained here is directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal. However, antibodies directed against non-polypeptide antigens (such as antigens associated with glycolipid tumors; see US Patent No. 5,091,178) are also contemplated. Where the antigen is a polypeptide, it can be a transmembrane molecule (for example, recipient) or ligand such as a growth factor. Exemplary antigens include those proteins described in section (3) below. Exemplary molecular targets for the antibodies included by the present invention include CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD22 and CD34; members of the ErbB receptor family such as the EGFR, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, MAC1, p1 50.95, VLA-4, ICAM-1, VCAM and av / B3 integrin including the a or β subunits of these (for example, anti-CD11a, anti-CD18 antibodies or anti-CD11b); growth factors such as VEGF; IgE; blood group antigens; flk2 / flt3 receptor, obesity receptor (OB); mpl receiver; CTLA-4; protein C, or any of the other antigens mentioned here.
[000146] Soluble antigens or fragments thereof, optionally conjugated with other molecules, can be used as immunogens for the generation of antibodies. For transmembrane molecules, such as receptors, their fragments (for example, the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells that express the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (for example, cancer cell lines) or they can be cells that have been transformed by recombinant techniques to express the transmembrane molecule.
[000147] Other antigens and their useful forms for the preparation of antibodies will be evident to those in the art. (ii) Polyclonal Antibodies
[000148] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the antigen to a protein that is immunogenic in the species to be immunized, for example, limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, ester of maleimidobenzoyl sulfosuccinimide (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N = C = NR, where R and R1 are different alkyl groups.
[000149] Animals are immunized against the antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injection of the solution intradermally in multiple locations. One month later, the animals are boosted with 1/5 of the {fraction (1/10)} of the original amount of antigen or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is analyzed for antibody titer. The animals are reinforced up to the titling plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and / or through a different cross-linking reagent. Conjugates can also be prepared in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. (iii) Monoclonal Antibodies
[000150] Monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be produced by recombinant DNA methods (US Patent No. 4,816,567).
[000151] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or simian monkey, is immunized as described above to extract the lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principies and Practice, pp. 59-103 (Academic Press, 1986)).
[000152] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused myeloma cells. For example, if myeloma cells of origin lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), whose substances prevent the growth of deficient cells of HGPRT.
[000153] Preferred myeloma cells are those that fuse efficiently, support the production of a high stable level of antibody by the selected antibody-producing cells, and are sensitive to a medium such as the HAT medium. Among these, the preferred myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11 available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[000154] The culture medium in which the hybridoma cells are growing is evaluated with respect to the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
[000155] After the hybridoma cells are identified that produce antibodies of the specificity, affinity and / or desired activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59 - 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as tumors in ascites in an animal.
[000156] Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, Protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Preferably, the Protein A affinity chromatography procedure using a pH gradient described herein is used.
[000157] The DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to the genes encoding the monoclonal antibody heavy and light chains) . Hybridoma cells serve as a preferred source of such DNA. Once isolated, DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise they do not produce the immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
[000158] DNA can also be modified, for example, by replacing the coding sequence for human heavy and light chain constant domains instead of homologous murine sequences (US Patent No. 4,816,567; Morrison, et al. , Proc. Natl Acad. Sci. USA, 81: 6851 (1984)), or by covalently joining the total or partial immunoglobulin coding sequence of the coding sequence for a non-immunoglobulin polypeptide.
[000159] Typically such non-immunoglobulin polypeptides are replaced by the constant domains of an antibody, or they are replaced by the variable domains of an antibody antigen combining site to create a chimeric bivalent antibody comprising an antigen combining site having specificity for an antigen and another antigen combination site having specificity for a different antigen.
[000160] Monoclonal antibodies can be isolated from phage antibody libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581597 (1991) describe the isolation of murine and human antibodies, respectively, using the phage libraries . Subsequent publications describe the production of high-affinity human antibodies (nM range) by chain rearrangement (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for building very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional hybridoma techniques for the isolation of monoclonal antibodies. (iv) Humanized and Human Antibodies
[000161] A humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from a "import" variable domain. Humanization can be essentially performed following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al. , Science, 239: 1534-1536 (1988)), by replacing the sequences of rodent CDRs or CDRs with the corresponding sequences of a human antibody. Consequently, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.
[000162] The choice of human variable domains, both light and heavy, to be used in the manufacture of humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993)). Another method uses a particular structure derived from the consensus sequence of all human antibodies in a particular subgroup of light or heavy chains. The same structure can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immnol., 151: 2623 (1993) ).
[000163] It is also important that the antibodies are humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this objective, according to a preferred method, humanized antibodies are prepared by a process of analysis of the source sequences and several conceptual humanized products use three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and show likely conformational three-dimensional structures of selected candidate immunoglobulin sequences. The inspection of these presentations allows the analysis of the probable role of residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, RF residues can be selected and combined from the recipient and the import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, CDR residues are directly and more substantially involved in influencing antigen binding.
[000164] Alternatively, it is now possible to produce transgenic animals (for example, mice) that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that homozygous deletion of the antibody heavy chain binding region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the immunoglobulin genetic disposition from the human germline in such germline mutant mice will result in the production of human antibodies after the antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355: 258 (1992). Human antibodies can also be derived from the phage display libraries (Hoogenboom et al., J. Mol. Biol, 227: 381 (1991); Marks et al., J. Mol. Biol, 222: 581-597 (1991) ; Vaughan et al. Nature Biotech 14: 309 (1996)). (V) Antibody Fragments
[000165] Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived through proteolytic digestion of intact antibodies (see, for example, Morimoto et al. Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985 )). However, these fragments can now be produced directly by the recombinant host cells. For example, antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from the culture of the recombinant host cell. A single chain Fv fragment (scFv) can also be isolated. See WO 93/16185. Other techniques for the production of antibody fragments will be evident to the skilled practitioner. (vi) Multispecific Antibodies
[000166] Multispecific antibodies have binding specificities for at least two different antigens. Although such molecules normally bind only two antigens (i.e., bispecific antibodies, BsAbs), antibodies with additional specificities such as triespecific antibodies are included by this expression when used herein.
[000167] Methods for producing bispecific antibodies are known in the art. Traditional production of bispecific full-length antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983 )). Because of the random variety of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is quite complicated, and product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).
[000168] According to another method described in WO 96/27011, the interface between a pair of antibody molecules can be prepared to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (for example, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (for example, alanine or threonine). This provides a mechanism to increase the yield of the heterodimer over other unwanted end products such as homodimers.
[000169] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have, for example, been proposed to target cells of the immune system to unwanted cells (US Patent No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03,089 ). Heteroconjugate antibodies can be produced using any convenient cross-linking method. Suitable crosslinking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with various crosslinking techniques.
[000170] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the sodium arsenite of the dithiol complex agent to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfide. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
[000171] Recent progress has facilitated the direct recovery of E. coli Fab'-SH fragments, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody molecule F (ab ') 2. Each Fab 'fragment was separately secreted from E. coli and subjected to targeted chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells that overexpress the ErbB2 receptor and normal human T cells, as well as to activate the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
[000172] Various techniques for the production and isolation of bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies were produced using leucine zippers. Kostelny et al., J. Immunol, 148 (5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab 'parts of two different antibodies by genetic fusion. The antibody homodimers were reduced in the hinge region to form monomers and then oxidized again to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for the preparation of bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Consequently, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Another strategy for the preparation of bispecific antibody fragments through the use of single chain Fv dimers (sFv) has also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994). Alternatively, the antibodies can be "linear antibodies" as described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH -CH1- VH and VL) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.
[000173] Antibodies with more than two valences are contemplated. For example, specific antibodies can be prepared. Tutt et al. J. Immunol 147: 60 (1991). Immunoadhesins
[000174] The simplest and most constant immunoadhesin design combines the adhesin binding domain (for example, the extracellular domain (ECD) of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the immunoadhesins of the present invention, the nucleic acid encoding the adhesin binding domain will be fused at the C-terminal to the nucleic acid encoding the N-terminal of an immunoglobulin constant domain sequence, however, the N-terminal mergers are also possible.
[000175] Typically, in such fusions the encoded chimeric polypeptide will at least functionally retain the CH2 and CH3 domains of the active hinge of the immunoglobulin heavy chain constant region. Fusions are also prepared for the C-terminus of the Fc part of a constant domain, or immediately the N-terminus for the CH1 of the heavy chain or the corresponding region of the light chain. The precise location where the merger takes place is not critical; the particular sites are well known and can be selected in order to optimize the biological activity, secretion or binding characteristics of the immunoadhesin.
[000176] In some embodiments, the adhesin sequence is fused at the N-terminus of the immunoglobulin G1 Fc domain (Ig G1). It is possible to fuse the entire heavy chain constant region into the adhesin sequence. Preferably, however, a sequence that begins at the hinge region just upstream of the papain cleavage site that chemically defines IgG Fc (i.e., residue 216, taking the first residue from the constant region of the heavy chain to be 114) , or analogous sites of other immunoglobulins are used in the fusion. In some embodiments, the adhesin amino acid sequence is fused to (a) the hinge region and either CH2 and CH3 or (b) the CH1, hinge, CH2 and CH3 domains, of an IgG heavy chain.
[000177] For bispecific immunoadhesins, immunoadhesins are assembled as multimers, and particularly as heterodimers or heterotetramers. Generally, these assembled immunoglobulins will have known unitary structures. A basic four-chain structural unit is the way in which IgG, IgD and IgE exist. A four-chain unit is repeated on the highest molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in the multimeric form in the serum. In the case of a multimer, each of the four units can be the same or different.
[000178] Several exemplary assembled immunoadhesins within the scope of this document are schematically diagrammed below: (a) ACL-ACL; (b) ACH- (ACH, ACL-ACH, ACL-VHCH, or VLCL-ACH); (c) ACL-ACH- (ACL-ACH, ACL-VHCH, VLCL-ACH, or VLCL-VHCH) (d) ACL-VHCH - (ACH, or ACL-VHCH, or VLCL-ACH); (e) VLCL-ACH - (ACL-VH Ch, or VLCL-ACH); and (f) (A-Y) n- (VLCL-VHCH) 2, (g) that each A represents the same or different adhesin amino acid sequences; VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; Immunoglobulin CH; is a heavy chain constant domain of n is an integer greater than 1; Y designates the residue of a covalent cross-linking agent.
[000179] For the sake of brevity, the previous structures only show key characteristics; they do not indicate the union (J) or other immunoglobulin domains, nor are disulfide bonds shown. However, where such domains are required for binding activity, they must be constructed to be present in the normal locations that they occupy on immunoglobulin molecules.
[000180] Alternatively, the adhesin sequences can be inserted between the immunoglobulin heavy chain and the light chain sequences, such that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the adhesin sequences are fused at the 3 'end of an immunoglobulin heavy chain in each branch of an immunoglobulin, between the hinge and the CH2 domain, or between the CH2 and CH3 domains. Similar constructions have been reported by Hoogenboom, et al., Mol. Immunol. 28: 1027-1037 (1991).
[000181] Although the presence of an immunoglobulin light chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain can be present covalently associated with an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused with adhesin . In the first case, DNA encoding an immunoglobulin light chain is typically co-expressed with DNA encoding the adhesin-immunoglobulin heavy chain fusion protein. After secretion, the hybrid heavy chain and the light chain will be covalently linked to provide a structure such as immunoglobulin comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. Suitable methods for preparing such structures are, for example, disclosed in US Patent No. 4,816,567, issued March 28, 1989.
[000182] Immunoadhesins are most conveniently constructed by fusing the cDNA sequence that encodes the part in the structure into an immunoglobulin cDNA sequence. However, fusion of genomic immunoglobulin fragments can also be used (see, for example, Aruffo et al., Cell 61: 1303-1313 (1990); and Stamenkovic et al., Cell 66: 1133-1144 (1991) ). The latter type of fusion requires the presence of Ig regulatory sequences for expression. CDNAs encoding IgG heavy chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction techniques ( PCR). The cDNAs encoding the "adhesin" and the immunoglobulin parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in selected host cells. Other Polypeptides Containing the CH2 / CH3 Region
[000183] The polypeptide to be purified is one that is fused to, or conjugated to, a CH2 / CH3 region. Such fusion polypeptides can be produced so as to increase the serum half-life of the protein and / or facilitate the purification of the protein by Protein A affinity chromatography. Examples of biologically important proteins that can be conjugated in this way include renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin chain A; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIc, factor IX, tissue factor and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; alpha and beta factor of tumor necrosis; encephalinase; RANTES (regulated on the T cell of expressed and secreted normal activation); inflammatory human macrophage protein (MIP-1-alpha); a serum albumin such as human serum albumin;
[000184] Muellerian inhibiting substance; relaxin chain A; relaxin chain B; prelaxin; peptide associated with mouse gonadotropin; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin, activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; Protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a factor growth such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II and -II (IGF-I and IGF-II); des (1-3) -IGF-I (cerebral IGF-I), are insulin-like growth factor binding proteins; CD proteins, such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -β, and - Y; colony stimulating factors (CSFs), for example, M-CSF, GM-CSF, and G-CSF; interleukins (ILs), for example, IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; deterioration accelerating factor; viral antigen such as, for example, part of the AIDS envelope; transport proteins; resident receivers; adressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor-associated antigen such as the EGFR, HER2, HER3 or HER4 receptor; and fragments of any of the polypeptides listed above. Polypeptide Expression
[000185] The polypeptide to be purified using the method described herein is generally produced using recombinant techniques. The polypeptide can also be produced by the synthesis of peptides (or other synthetic means) or isolated from a native source.
[000186] For the recombinant production of the polypeptide, the nucleic acid that it encodes is isolated and inserted into a replicable vector for another cloning (amplification of the DNA) or for expression. The DNA encoding the polypeptide is easily isolated and sequenced using conventional procedures (for example, where the polypeptide is an antibody using oligonucleotide probes that are able to specifically bind to the genes encoding the antibody's heavy and light chains) . Many vectors are available. The components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence ( for example, as described in US Patent No. 5,534,615, specifically incorporated herein by reference).
[000187] The host cells suitable for cloning or expressing DNA in the vectors here are prokaryotic, yeast, or higher eukaryotic cells. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium , Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (for example, B. licheniformis 41P disclosed in DD 266,710 published on April 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.
[000188] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for polypeptide coding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species and strains are commonly available and useful here, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906 ), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);
[000189] Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
[000190] Host cells suitable for the expression of glycosylated polypeptide are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used here as the virus according to the present invention, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunia, tomatoes and tobacco can also be used as hosts.
[000191] However, interest has been greater in vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include, but are not limited to, SV40 monkey kidney cells transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary / DHFR cells (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2).
[000192] Host cells are transformed with the expression described above or cloning vectors for polypeptide production and cultured in conventional modified nutrient media as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences.
[000193] The host cells used to produce the polypeptide used in the methods of this invention can be grown in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), Dulbecco’s Modified Eagle’s Medium ((DMEM), Sigma) are suitable for host cell culture. In addition, any of the means described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US Patent to Re. 30,985, can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the medicine GENTAMYCIN ™), trace elements (defined as inorganic compounds generally present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements can also be included in the appropriate concentrations that should be known to those skilled in the art. Culture conditions, such as temperature, pH, and more, are those previously used with the host cell selected for expression, and will be evident to the artisan commonly versed.
[000194] When using recombinant techniques, the polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide is produced intracellularly, as a first step, particulate residues, host cells or lysed cells (for example, resulting from homogenization), are removed, for example, by centrifugation or ultrafiltration. Where the polypeptide is secreted into the medium, supernatants from such expression systems are generally primarily concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Compositions Including Pharmaceutical Formulations Comprising Polypeptides
[000195] The invention also includes compositions, such as pharmaceutical formulations. A pharmaceutical formulation comprising the polypeptide purified by the methods of the present invention, optionally conjugated to a heterologous molecule, can be prepared by mixing the polypeptide having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 21st edition (2005 )), in the form of lyophilized formulations or aqueous solutions.
[000196] The polypeptide product comprising a CH2 / CH3 region purified by the methods described herein can have the desired degree of purity of at least about 98% monomer or at least about 99% monomer.
[000197] "Pharmaceutically acceptable" carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
[000198] cyclohexanol; 3-pentanol, and m-cresol); low molecular weight polypeptide (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, Zn-protein complexes); and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).
[000199] Here the formulation can also contain more than one active compound as needed for the particular indication being treated, preferably those with complementary activities that do not negatively affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.
[000200] The active ingredients can also be captured in a microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and poly (methylmethacrylate) microcapsule, respectively, in release systems. colloidal medications (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 21st edition (2005).
[000201] The formulation to be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.
[000202] Controlled release preparations can be prepared. Suitable examples of controlled release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide variant, whose matrices are in the form of molded articles, for example, films, or microcapsules. Examples of controlled release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl methacrylate), or poly (vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl Y -L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid and leuprolide acetate), and poly-acid D - (-) - 3-hydroxybutyric.
[000203] The purified polypeptide as disclosed herein or the composition comprising the polypeptide and a pharmaceutically acceptable carrier is then used for various diagnostic, therapeutic or other uses known for such polypeptides and compositions. For example, the polypeptide can be used to treat a disorder in a mammal by administering a therapeutically effective amount of the polypeptide to the mammal.
[000204] The following examples are provided to illustrate, but not limit, the invention. EXAMPLES
[000205] It is understood that the examples and embodiments described here are for illustrative purposes only and that several modifications or changes in their understanding will be suggested to people skilled in the art and should be included within the spirit and scope of this application . Example 1: Chromatography of pH Gradient Elution Protein A
[000206] Six different proteins containing the CH2 / CH3 regions, anti-VEGF # 1 antibody, anti-CD20 antibody, anti-VEGF # 2 antibody, anti-MUC16 antibody, anti-CD4 antibody, and a branched anti-Met antibody were purified using a pH step gradient elution method. The method is described in Table 1. Table 1
Experimental Procedures
[000207] A Unicorn method file was built using the protein loading and buffer / pH composition parameters as listed in Table 1. This file was executed by a GE Healthcare AKTA (GE Healthcare) Explorer FPLC system (liquid protein chromatography) fast). The FPLC was a bench-scale instrument produced from plastic tubes and pumps that simulated a manufacturing purification process. The FPLC produced a "pH gradient" phase by mixing two buffers at a programmed rate of change using a two pump system (pump A and pump B) where the flow rate was maintained and the percentage of the flow of each of the delivered pumps changed. In the Unicorn program, a "% B" was designated in another through a certain volume (number of column volumes).
[000208] In the column equilibration phase, the column was removed from the storage solution as listed in Table 1 and prepared to load the protein materials. In the protein loading phase, the HCCF (harvested cell culture fluid) was pre-filtered using a 0.2 micron pore size vacuum filter and loaded onto the Protein A column (MABSELECT ™, MABSELECT SURE ™, POROS® MABCAPTURE ™ A, PROSEP® Va, or PROSEP® Ultra Plus). The proteins were loaded at a density in the range of 14 to 37 g / L. Most operations were performed at 21 g / L. In the Wash 1 phase, the Wash 1 buffer was used to push any load left on the Akta lines in the column. In the Wash 2 phase, impurities such as CHOP (Chinese Hamster Ovary Protein) were removed by the Wash 2 buffer. In the Wash 3 phase, the Wash 3 buffer was used to remove the Wash 2 buffer and the associated impurities from the Wash 2 buffer. column for the preparation of the elution phase. In the gradient elution phase of the pH step, a gradient formed by the precise manipulation of two pH buffers of different pHs by a two pump system was used to gradually move from a pH mixture of the two buffers to another mixture, defined in percentages . The elution parameter of "35-60% B" correlates with a pH gradient range of about 4.3 to 3.7. More specifically, 35% B corresponds to the pH of the elution buffer of 4.34, buffer composition of 16.25 mM acetate, 8.75 mM formate, and buffer conductivity of 1039 uS / cm. 60% B corresponds to an elution buffer pH of 3.69, buffer composition of 10 mM acetate and 15 mm formate, and buffer conductivity of 763 uS / cm. During this elution phase of most operations, fractions were taken throughout the elution and evaluated using a size exclusion HPLC assay to determine the monomer elution behaviors versus size variant (HMWS (High Molecular Weight Species) ), dimer, or fragment). These fractions were also subjected to a CHOP assay for the selected operation, and all fractions were measured for protein concentration using a NanoDrop UV spectrophotometer (Thermo Fischer Scientific, Wilmington, DE). In the regeneration phase, regeneration buffer was used to wash away any tightly bound impurities or excess product to minimize the transition between operations. In the storage phase, the Protein A column was removed from the regeneration buffer and stored in a solution that was designed to maintain the integrity of the column over time in disuse.
[000209] Phase lengths and flow rates were measured in CVs and centimeters per hour, respectively. The flow rate was sized by centimeters per hour (dividing the flow rate in cm / h by the height of the column bed in cm to reach column volumes per hour, also a standard unit), due to pressure concerns.
[000210] Chromatograms were collected and analyzed by the AKTA purification system (GE Healthcare) FPLC and its associated Unicorn software package. After the column A purification operation was performed, the UV, pH and conductivity absorbance traces (as well as other measured values or program instructions / logbook) were accessed and examined. The. Size Exclusion Chromatography Assay (SEC)
[000211] An analytical size exclusion chromatography (SEC) assay was performed on an Agilent 1200 series HPLC (Agilent Technologies, USA, part G1329A) and used to determine the relative levels of high molecular weight (HMWS) species, dimer , monomer and fragment for the collected samples. A 14.24 ml TSK G3000SWXL column, 7.8 mmD x 300 mmH (Tosoh Bioscience, Tokyo, Japan, part 08541) was used. Each sample was diluted in approximately 0.5 g / l of antibody using the potassium phosphate / potassium chloride HPLC operating buffer or sample injections were modified to standardize the mass loaded on the test column. All samples were prepared in 1.5 ml Agilent HPLC glass vials. The operations were 30 minutes with a flow rate of 0.5 ml / min. Sample injections were adjusted so that approximately 25 ng of antibody was loaded per sample. Sketches containing test buffers from the respective samples were operated on with each set of samples. 280 nm UV absorbance curves were analyzed manually using ChemStation (Agilent Technologies) or automatically using CHROMELEON® software (DIONEX, Sunnyvale, CA) to integrate the peaks separately to obtain the percent species values for the samples. The percentage values obtained in this test can be multiplied by the concentration (mg / ml) of the fraction to obtain an actual concentration or mass for each variant species of size in the sample (for example, SEC result: 4% HMWS, 3% dimer, 92% monomer, 1% fragment; sample concentration: 2 g / L; sample volume: 10 ml; 1.84 g / L monomer, 18.4 mg of total monomer in the sample). B. CHOP Assay
[000212] The samples from selected operations were subjected to a test group that performed a standard enzyme-linked immunosorbent assay (ELISA) and validated to quantify CHOP levels. Affinity purified goat anti-CHOP antibodies were immobilized in microtiter plate wells. Dilutions of samples containing CHOP, standards and controls, were incubated in the reservoirs, followed by incubation with goat anti-CHOP antibodies conjugated to horseradish peroxidase. The enzymatic activity of horseradish peroxidase was detected with o-phenylenediamine dihydrochloride. CHOP was quantified by reading the absorbance at 492 nm in a microtiter plate reader. A curve fitting computer program was used to generate the standard curve and automatically calculate the sample concentration. The test range for the ELISA was typically 5 ng / ml to 320 ng / ml. The results were standardized in ppm for comparisons together. Results of
[000213] The way of eluting a step gradient is shown in Figure 1. All proteins were eluted until the end of the pH decline. A larger portion of the proteins were eluted from the column more quickly, but a lot of protein remained in the column and were eluted during the gradient. At the lowest pH, the separation between the desired product (elution range around pH 4.6 to 3.7 with slight variation from molecule to molecule) and the undesirable aggregate (elution range around pH 3.9 to 3.5) occurred.
[000214] In all six protein molecules tested (anti-VEGF antibody # 1, anti-CD20 antibody, anti-VEGF antibody # 2, anti-MUC16 antibody, anti-CD4 antibody, and a branch branch anti-Met antibody ), high percentages (~ 100%) of monomer were observed in the initial gradient fractions and the final residual part of the gradient contained much higher levels of aggregate species (> 50%). These SEC results are shown in Figures 2-4B. Since this series of tested molecules encompasses the larger classes of protein molecules (for example, chimeric antibodies, thioMabs (recombinant monoclonal antibodies having a point mutation by replacing an amino acid residue with cysteine), IgG4s, and antibody fragments produced by E. coli), this pH step gradient method suggests wide applicability for all polypeptides / proteins containing the CH2 / CH3 region (eg, Fc region). Example 2: CHOP separation for anti-CD20 antibody, anti-VEGF antibody # 1, and anti-MUC16 antibody on a Protein A chromatography column using standard step elution and gradient elution from the pH step
[000215] Using the pH step gradient elution protein method as described in Example 1, anti-CD20 antibody levels per fraction in mg / ml (from the off-line UV 280 absorbance at the top of the bench, which scanned with the AKTA / Unicorn UV 280 readings in line for the gradient elution phase of the step as seen in the anti-VEGF antibody chromatogram # 1 in Figure 1A) and the CHOP levels per fraction in ppm were measured. As seen in the left panel of Figure 5 (anti-CD20 antibody and CHOP elution), the lower pH later fractions of the H step gradient elution contained very little anti-CD20 antibody compared to the amount of CHOP. The data from the control operation done at the same time as the elution of pH gradient from the anti-CD20 antibody step are shown in the top row of the table in Figure 5. The control operation was using the same conditions as described in Table 1, except that no gradient of the pH step was used during the protein elution phase, and the protein was eluted at pH 3.6 or below. The bottom line shows a small, but acceptable, decrease in anti-CD20 antibody yield using the pH step gradient, (note: this loss of yield due to aggregate removal is expected; yield is calculated using the value of HCCF titration that includes aggregates in the total amount of product loaded on the column). About 5% less aggregates and half of the CHOP levels were observed compared to the control set. The results establish an unexpected benefit of increased purity using the gradient elution of the pH step.
[000216] CHOP separations for both anti-VEGF antibody # 1 and anti-MUC16 antibody were also performed using the pH step gradient method described in Example 1. Similar to the pattern seen in the CHOP antibody graph anti-CD20, more CHOP was eluted at the end of the pH gradient in proportion to the elution of the anti-VEGF antibody, indicating that the gradient elution of the pH step separated the impurities from the host cell into this protein molecule, as well as the antibody anti-CD20. See Figure 6. For CHOP separation in the anti-MUC16 antibody, this antibody had much higher CHOP levels compared to the elution pattern seen in the anti-VEGF #l antibody. See Figure 7. Consequently, a significant amount of CHOP can be fractionated from the anti-MUC16 antibody using the pH step gradient method as described above. Example 3: The clearance of virus particles using pH gradient elution protein A chromatography
[000217] Using the pH gradient elution protein A chromatography method as described in Example 1, the clearance of the anti-VEGF # 1 antibody virus particle was measured. Chromatography of protein A eluting the pH gradient of the step
[000218] All phases and buffers were the same as those used in Example 1. Fractions were tested for retrovirus-like particle counts using a quantitative polymerase chain reaction assay. The. Retrovirus-like Quantitative Particle Polymerase Chain Reaction Assay (RVLP QPCR)
[000219] The RVLP endogenous virus particle assay is a quantitative real-time PCR assay. Viral RNA was extracted from the samples using Qiagen EZ1 (Qiagen, Valencia, CA). Sample sizes were 0.4 ml (undiluted and 1:10 diluted HCCF, undiluted protein A set). The extraction efficiency was confirmed by including a reference standard HCCF sample with a known CHO retrovirus particle titer. Genomic DNA was removed by DNase digestions by treating the extraction eluate with 0.2 units / ml DNase I at an elevated temperature for 30 min. DNase was then inactivated by heat at 70 ° C for 15 min. The absence of retroviral DNA was confirmed by analyzing the samples without reverse transcriptase.
[000220] Quantitative real-time PCR assays to measure CHO retrovirus genomes were performed as described in De Wit et al. (Biologicals, 28 (3): 137-48 (2000)), but with the use of a new probe in the nearby region. Reagents and procedures have also been updated and improved. The primers and probe sequences were designed to amplify a fragment in the highly conserved pol region of the CHO type C retrovirus genome. Each retrovirus-like particle contains two genomic RNA molecules. Oligonucleotide probes and primers were ordered from Applied Biosystems (Foster City, CA) and Invitrogen (Carlsbad, CA). Viral clearance was expressed as a log 10 reduction value, or LRV, which is the difference of log 10 (total virus) in the protein load (HCCF) and in the product set. The total virus was obtained from virus titers (particles / ml or nU / ml) in the samples and sample volume (ml). Figure 18 shows the endogenous virus-like particle count for each fraction taken from the gradient elution of the pH step. Some viruses eluted at the beginning of the gradient at the largest peak, but a larger portion of the virus eluted at the end of the elution residue. Consequently, the separation of these product RVLPs can benefit the pH elution gradient or Protein A chromatography of the total gradient elution of overall efficiency in terms of viral clearance. Figure 19 shows the LRV for each fraction compared to the HCCF load. The graph was based on a calculation using the values in Figure 18. A large decrease in LRV was observed in the elution fractions rich in aggregate afterwards. The LRV was high (desirable effect) in the median elution, higher monomeric polypeptide fractions. Example 4: Purification of basic polypeptide variants using pH gradient eluting protein A chromatography
[000221] Using the pH step gradient elution protein A chromatography method as described in Example 1, the clearance of basic polypeptide variants (or basic variants) of anti-VEGF antibody # 1 was measured. PH gradient elution protein A chromatography step
[000222] All phases and buffers were the same as those used in Example 1. The fractions were subjected to the ion exchange variant test. The. Ion Exchange Variant Assay
[000223] An analytical ion exchange chromatography (IEC) assay was performed on an Agilent 1200 series HPLC (Agilent Technologies, USA, part G1329A) and used to determine the relative peak peak levels of acidic and basic charged variants for samples of anti-VEGF antibody # 1 collected. A Dionex ProPac WCX-10 column, 4.6 x 250 mm (Dionex product no. 054993) was used with a gradient of ACES [N- (2-acetamido) -2-aminoethanesulfonic acid] and NaCl under high temperature conditions. Sample preparation included buffer exchange samples in the IEC mobile phase prior to a 20-minute heated digestion with Carboxypeptidase (CpB). Approximately 50 µg of anti-VEGF # 1 antibody was injected into the column per sample. 280 nm UV traces were obtained and integrated using ChemStation software (Agilent Technologies). The percentages of integration for each category of acidic, basic and main peak species were analyzed with respect to the tendencies of ionic variant composition composition through the gradient. Figure 17 shows the result of the peak integration test of the ion exchange variant through 20 fractions of the gradient elution from the pH step of Protein A. The percentage of basic variants present in the fractions increases dramatically in the residual part of the gradient elution from the step of Protein A pH. This residual part of the gradient elution is the same part where increased CHOP and aggregation separation were observed, as described in Examples 1 and 2. Example 5: Gradient elution of the pH step using multiple chromatography columns of Protein A
[000224] Both MABSELECT SURE ™ and MABSELECT ™ resins were tested for the pH step gradient elution method as described above. These two Protein A resins, although similar in name, have different affinity binders incorporated. MABSELECT ™ carries the native Protein A ligand, which binds to the Fc parts of the antibodies. MABSELECT SURE ™ carries a modified form of Protein A that has been chemically altered to be stable in high pH solutions for short amounts of time. The hidden Akta elution profiles show that the elution traces are extremely similar for the two Protein A resins. As can be seen from the SEC integration profile for MabSelect in Figure 8, comparable aggregate separation was achieved using this resin. Other resins successfully tested for aggregate separation were PROSEP® Va, PROSEP® Ultra Plus, and POROS® MABCAPTURE ™ A. Consequently, several affinity resins (eg, MABSELECT ™, MABSELECT SUERE ™, PROSEP® Va, PROSEP®® Ultra Plus, and POROS® MABCAPTURE ™) can be used in the gradient elution method of the pH step for fractional impurities. Example 6: Design of experiments (DOE) using various parameters for the pH gradient elution method
[000225] Several parameters that are important in most chromatography processes have been explored with respect to anti-VEGF antibody # 1 using an operation study 35 statistically designed within the ranges indicated in Table 2. The parameter "initial elution% B" affects the starting pH of the elution phase (which plays an important role in determining the shape of the elution curve. The higher the starting% B, the lower the starting elution pH, the more the protein eluted from the column in the first fractions), as well as the slope of the global gradient. The parameters were varied simultaneously in a fractional factorial study designed to elucidate the main effects as well as the interactions. All operations were fractionated during elution, and the aggregate and concentration were evaluated for all fractions. An inserted calculation was used to determine the levels of the simulated set monomer that should result in exactly 85% yield (the lower limit of the step yield target) and these values were used to compare the effectiveness of each series of performance parameters. operation in separating monomer from aggregate efficiently. Table 2 - DOE Parameter Ranges
* All elutions ended at 60% B (pH 3.7) Experimental Procedures
[000226] All parameter changes with the exception of the column bed height (which requires the storage of several columns) were examined using the Unicorn software. CV fractions were taken throughout all elutions, evaluated using the HPLC SEC (as described above), and measured for protein concentration (UV absorbance 280 as measured on a NanoDrop UV spectrophotometer). In order to better standardize the results for comparison, data from several series of fractions were compiled and a calculation was used to interpolate the overall yield and the SEC profile of a set of each of the operations.
[000227] The JMP® software package (SAS, Cary, NC) was used to generate an operation plan for exploiting the fractional factorial parameter. One of the operations was selected as the "exemplary manufacturing operation" in the series due to its high throughput, high monomer level, and low set size (Figure 11). Results of
[000228] The Pareto chart of the DOE results shows that the "% B starting" is the most influential parameter in determining aggregate separation, followed by load density (lower is better) and residence time (residence time) was calculated by dividing the controlled parameters of the bed height (cm / hp) by the flow rate (cm / h) for h / hp. Consequently, the lower the starting% B, the higher the starting elution pH , the more efficient the separation of aggregates, the lower the density of the load, the more efficient the separation of aggregates, and the longer the residence time, the more efficient the separation of the aggregate. , the interaction profiles of this study show that there was an interaction between the load density parameters and B% starting, as well as an interaction between the residence time and the load density. departure had a greater effect on the monomer levels of simulated sets with 85% yield than if the operation were done at lower load densities. In addition, in the increased charge density, the residence time played a much greater role in the gradient's ability to fractionate the aggregates more efficiently. A lower rate can be used during elution to achieve high product yield with the pH step gradient method. See Figure 10. Figure 11 shows that the set for this operation was lower than 10 CVs (for example, 5.4 CV or 6 CV) while releasing less than 1% of aggregate with more than 85% yield.
[000229] In addition to the main effects (Pareto chart in Figure 9), interactions (Figure 10), and exemplary fabrication operation (Figure 11), it was observed that the overall elution length had no effect on the separation of aggregates, but it can be manipulated to decrease the size of the set to result in a set that has high purity and high yield, but in a smaller volume (which is preferable for the manufacturing scale). Likewise, the use of the pH step gradient resulted in smaller set volumes with purity almost comparable to that given by the complete gradient (see Example 8). The pH step gradient was also found to be robust in several process changes, including minor changes in charge density and flow rate, as well as not being completely affected by bed height. Example 7: Protein purification using ion exchange membrane chromatography following the pH step gradient Protein A chromatography
[000230] To determine whether downstream column chromatographies can be eliminated from the purification process or replaced with membranes, the bench scale cycle of the Protein A pH step gradient over anti-VEGF antibody # 1 (with a less than 1% aggregate and a high yield) was loaded downstream of the loaded membranes. MUSTANG® S (Pall Corporation) and MUSTANG® Q (Pall Corporation) membranes represent a cation exchange membrane and an anion exchange membrane, respectively. Success was measured by the membrane's ability to achieve the same overall purities and yields compared to the typical downstream column process. Experimental Procedures
[000231] The parameters used to determine the ideal load conditioning for CHOP and the aggregate clearance on the S and / or Q membranes were taken from previous studies on the ideal load conditioning for the CHOP clearance on the S and / membrane. or Q using a standard Protein A step set in Tables 3A and 3B. In these previous studies, promising results were shown when a standard Protein A step set was used (control group, using no pH gradient and protein elution at pH 3.6 or below); however, this process had slightly higher than desired CHOP levels and did not clear any aggregates from the process. In these previous studies, the membranes were loaded in 5 kg / L of membrane and the ideal loading conditions were observed with respect to the removal of impurities. These same conditions were used for the gradient elution set of the Protein A step to compare the performance of the different Protein A sets on these membranes with the primary impurities oriented as CHOP and aggregate. Table 3A: Previous parameters for determining the optimal load conditioning for CHOP clearance on S or Q membranes
Table 3B: Previous parameters to determine whether orthogonal impurity clearance can be achieved by operating S and Q membranes in series.

[000232] In the initial studies, Protein A standard step elution sets were conditioned to specific pHs and conductivities and passed through cationic (MUSTANG® S) and anionic (MUSTANG Q) membrane units on a laboratory scale that were connected to the AKTA ™ FPLC purification system. Flow rates were maintained and traces of pressure were examined for evidence of fouling / deterioration of permeability. The fractions were taken at various charge densities and evaluated for CHOP (ELISA) and antibody concentrations (UV 280 absorbance in the NanoDrop UV spectrophotometer). The results of these tests were compared at various loading densities to find the ideal loading conditions for the final CHOP clearance. In the subsequent studies, the gradient elution set of the Protein A pH stage was conditioned as ideal observed in the initial studies. Thereafter, the performance of the step elution gradient set was compared to that of the standard protein A step elution in downstream membrane capacity in cleaning CHOP and other impurities, while maintaining high throughput and low levels of aggregation. Two sizes of cation exchange membrane and three sizes of anion exchange membrane were also used to test the reproducibility and scale representation of the results. Results of
[000233] When the Protein A step set was used as the filler for a series of cationic to anionic exchange membrane purification, the lowest CHOP levels obtained were 15 to 20 ppm for membranes loaded with 5 kg / L of membrane. Since the membranes did not clean the aggregate, the levels of this impurity were unacceptably high in the final assemblies. Conversely, when the gradient elution set of the pH step of protein A was loaded onto these same membranes, CHOP levels were 0 to 15 ppm and aggregate levels were still low in the final sets, showing a benefit for the overall process.
[000234] In addition, the final sets of each of the three combinations of ion exchange membranes tested resulted in final sets of high yield and high purity. In all cases, aggregate levels remained low through pH adjustment and membrane processing, and CHOP levels were also lower than that seen in previous control studies, showing an unexpected CHOP reduction benefit for use a lower aggregate protein A pool as a food. See Figure 12. Consequently, the use of ion exchange chromatography membranes in place of the usual ion exchange chromatography columns eliminates the need for many typical column chromatography buffers and other manufacturing time / space drawbacks. More importantly, since these charged membranes were used in the overload mode (that is, the charge is passed through the membrane at a high charge density with no washing or elution steps required to generate a highly purified assembly), the membranes carry take into account a continuous purification process downstream of the Protein A chromatography step of the pH gradient.
[000235] SEC integration profiles between the pilot scale (4.1 L) and the 28 mL bench scale are also shown to be extremely similar, indicating that the pH step gradient of Protein A can be successfully scaled and that any small-scale results can be considered indicative of performance on a larger scale. See Figure 13. These results indicate an unexpected benefit of reproducibility and scalability of the gradient elution Protein A chromatography of the pH step. Example 8: Parvovirus Filter Operations with VIRESOLVE® Pro A. Comparison of the deterioration of permeability with VIRESOLVE® Pro
[000236] The gradient of the Protein A pH step was compared to the standard Protein A step (control group, using no pH gradient and protein elution at pH 3.6 or below) in terms of facilitating greater mass yield on a parvovirus filter (VIRESOLVE® Pro, Millipore, Inc.). A standard HCCF food was used to run both the Protein A pH step gradient and the standard Protein A step control over QSFF (Q Sepharose Fast Flow (anion exchange column, GE Healthcare)) in a standard flow through the mode before operation on the VIRESOLVE® Pro parvovirus filter. Various operating conditions with VIRESOLVE® Pro were tested: 1) elution set from the standard Protein A step with the sterile inline SHC pre-filter, 2) elution of the standard Protein A step with a cation exchange membrane adsorber (CEX) as an in-line prefilter, 3) the gradient set of the Protein A pH step with the SHC prefilter (a sterile grade filter 0.2 micron unloaded), and 4) the Protein A pH step gradient set with a CEX membrane adsorbent as an in-line pre-filter. Some sets were operated with repetition. The VIRESOLVE® Pro was operated in a filtration configuration that uses a series of peristaltic pumps, scales, and pressure sensors to report the data in a spreadsheet.
[000237] The gradient set of the pH step with the sterile SHC in-line filter performed similarly in the step set with the cation exchange membrane, also presenting around a six-fold increase in the possible mass yield over the VIRESOLVE® Pro. See Figure 14. Consequently, this result indicates the unexpected benefit of the protein A pH step gradient in removing filter scale with VIRESOLVE® Pro without the aid of a CEX membrane pre-filter. Additionally, the performance of VIRESOLVE® Pro benefited from the combination of the CEX membrane adsorbent with the Protein A pH step gradient set, resulting in an unexpected approximate 18-fold improvement in potential mass yield compared to the set operation of standard step elution of Protein with SHC over VIRESOLVE® Pro. B. CHOP and SEC experiments with VIRESOLVE® Pro
[000238] The samples were taken at different points during the operation sequences with VIRESOLVE® Pro and analyzed for CHOP and SEC. The standard Protein A step sequence with SHC in line had very low CHOP levels, but still contained aggregates. The aggregate levels remained low in the gradient experiments of the Protein A pH step through different process steps and resulted in a final set with less than 1% aggregate. This result was a significant improvement over the set released by the set from the standard stage. In addition, the CHOP levels for the pH step gradient SHC-VIRESOLVE® Pro sets were comparable to the levels in the standard step operation with the cation exchange membrane. The levels were slightly lower than those of the standard step with the SHC. See Tables 4A and 4B. These results show that not only did the pH step gradient method produce higher mass yield compared to the standard step set, but it also produced comparable or better purity. Table 4
Table 4B
Example 9: Total pH gradient elution of Protein A over Protein A
[000239] Total pH gradient elution of Protein A was also tested. All phases and buffers were the same as those used in the pH step gradient method as described in Example 1, except that 35-60% B as used in the pH step gradient was decreased to 25-60% at the beginning of the gradient, which resulted in a larger initial pH gradient (starting around pH 4.6 and ending around pH 3.7). 25% B corresponds to the pH of the elution buffer of 4.58, buffer composition of 18.75 mM acetate and 6.25 mM formate, and buffer conductivity of 1141 uS / cm. 60% B corresponds to the elution buffer pH of 3.69, buffer composition of 10 mM acetate and 15 mM formate, and buffer conductivity of 763 uS / cm. See Table 5. Table 5

[000240] The way of eluting a total gradient is shown in Figure 15. All products were eluted by the end of the pH decline. The chromatogram for the total pH gradient is the same in all phases as the gradient for the pH step, except that the elution of the antibody starts at a higher pH. The lack of the initial high 280 UV tip at the beginning of the gradient resulted in lower concentration fractions at the beginning of the gradient, with more protein distributed throughout the rest of the elution phase. This took into account the greater separation of species that eluted preferentially at higher pHs than the monomer. Thus, this technique can be equally effective in cleaning impurities that separate from the desired product at lower pHs (ie, aggregates and CHOPs separated using the step gradient), with the only drawback being the final set volume in comparison with the gradient of the pH stage (lower fraction concentrations at the beginning of the gradient translate into a need to gather more fractions together to achieve a set of simulation of a desired yield). SEC traits and integrations for this total gradient also demonstrate the separation of monomer aggregates at lower pHs, suggesting that the benefits and applicability of the pH step gradient can be extended to a total gradient as well. Example 10: Formation of aggregates in the Protein A Study
[000241] To ensure that an aggregate was not formed during the low pH or the residual end of the pH step gradient elution and that the Protein A pH step gradient technique actually separated a monomer from an aggregate, instead To motivate the formation of an aggregate, purified materials were used to show that the aggregate levels of the food were not increased by processing on Protein A, with or without HCCF components. See Table 6. Table 6
* SEC monomer assay not used in HCCF

权利要求:
Claims (18)
[0001]
1. Method for purifying a polypeptide comprising a CH2 / CH3 region, characterized by the fact that it comprises the steps of: (a) attaching the polypeptide to Protein A; and (b) eluting the polypeptide with a pH gradient starting at 5.0 or less using an elution buffer, where the elution buffer contains a high pH buffer and a low pH buffer and where the pH gradient is formed by adjusting a percentage of each pH buffer in the elution buffer, where the high pH buffer has pH 5.0 and the low pH buffer has pH 2.7, where the pH gradient ends at 3.7, and in which an aggregate, a host cell impurity, a virus filter inlay, a virus particle and a virus-like particle are removed from the desired polypeptide.
[0002]
2. Method according to claim 1, characterized by the fact that the percentage of low pH buffer starts at 35%.
[0003]
Method according to claim 2, characterized in that the elution buffer containing the 35% low pH buffer comprises 16.25 mM acetate and 8.75 mM formate.
[0004]
4. Method according to claim 1, characterized by the fact that the percentage of low pH buffer starts at 25%.
[0005]
Method according to claim 4, characterized in that the elution buffer containing the 25% low pH buffer comprises 18.75 mM acetate and 6.25 mM formate.
[0006]
6. Method according to claim 1, characterized by the fact that the percentage of low pH buffer starts at 40%.
[0007]
Method according to claim 6, characterized in that the elution buffer containing the 40% low pH buffer comprises 15 mM acetate and 10 mM formate.
[0008]
8. Method according to claim 1, characterized by the fact that the pH gradient starts at pH 4.6.
[0009]
9. Method according to claim 1, characterized by the fact that the host cell impurity is Chinese Hamster Ovary Protein (CHOP).
[0010]
Method according to any one of claims 1 to 8, characterized in that a variant of basic polypeptide is separated from the polypeptide.
[0011]
Method according to any one of claims 1 to 10, characterized in that the polypeptide is an antibody.
[0012]
Method according to claim 10, characterized in that the antibody is a monoclonal antibody, a polyclonal antibody, a multi-specific antibody, or an antibody fragment.
[0013]
Method according to any one of claims 1 to 10, characterized in that the polypeptide is an immunoadhesion.
[0014]
Method according to any one of claims 1 to 13, characterized in that the polypeptide has a purity of at least 98% monomer.
[0015]
Method according to any one of claims 1 to 14, characterized in that the ratio of a host cell impurity to the purified polypeptide is at least about 20% lower than that of a purified polypeptide by a step elution method, wherein the step elution method comprises binding the polypeptide to Protein A and eluting with a pH starting at 3.6 or below.
[0016]
16. Method according to claim 15, characterized in that the ratio of a host cell impurity to the purified polypeptide is at least about 60% lower than that of a purified polypeptide by an elution method. stepwise, where the stepwise elution method comprises binding the polypeptide to Protein A and eluting with a pH starting at 3.6 or below.
[0017]
17. Method according to claim 15 or 16, characterized in that the purified polypeptide is a polypeptide monomer.
[0018]
18. Method according to any one of claims 1 to 17, characterized in that the purification is a process on a manufacturing scale.

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WO2011028753A1|2011-03-10|

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BR112012004697A2|2015-09-08|

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EP2473617A4|2014-04-09|

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BR112012004697B8|2021-05-25|

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HRP20200581T1|2020-07-10|

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JP2013503877A|2013-02-04|

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SI2473617T1|2020-07-31|

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KR101764449B1|2017-08-02|

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KR20120106719A|2012-09-26|

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CA2772653C|2019-06-25|

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CA2772653A1|2011-03-10|

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PL2473617T3|2020-06-29|

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US20130041139A1|2013-02-14|

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EP2473617A1|2012-07-11|

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MX2012002565A|2012-05-29|

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US9428548B2|2016-08-30|

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法律状态:
2018-10-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2019-11-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2019-12-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-06-02| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-12-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 09/02/2021, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/09/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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US23886709P| true| 2009-09-01|2009-09-01|

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US61/238,867|2009-09-01|

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