Purification methods for the removal of tyrosine sulfated antibody variants; purified compositions

Abstract

The present invention provides antibody and antigen-binding fragment compositions in which sulfated tyrosine variants in which one or more tyrosine residues in an immunoglobulin chain are sulfated have been removed and purified, and a method for purifying the same. [Solution] We provide a composition that does not contain sulfated tyrosine on CDR-L1 of an anti-LAG3 antibody having a specific sequence, and a method for removing tyrosine-sulfated antibodies or antigen-binding fragments thereof from an aqueous mixture containing tyrosine-sulfated and non-tyrosine-sulfated antibodies or antigen-binding fragments thereof, which method includes adjusting the pH of the mixture to about 6.5 to about 7.0, contacting the mixture with an anion exchange resin, and retaining the non-resin-bound aqueous fraction of the mixture from the resin.

Description

[Technical field]

[0001] The present invention relates to compositions comprising antibodies and antigen-binding fragments thereof that lack tyrosine sulfation. and purification methods for the preparation of the compositions. [Background technology]

[0002] Tyrosine sulfation occurs when a sulfate trioxide (SO3) group is attached to the amino acid tyrosine group. This PTM is a post-translational modification (PTM) that is covalently attached to a hydroxyl group on the ribosome. It occurs in the trans-Golgi network and is a tyrosylprotein sulfotransferase (TPST) The molecular mechanism is catalyzed by two enzymes: 3'-phosphoadenosine-5' -phosphosulfate to tyrosine, which is involved in the transfer of activated sulfate to various proteins. Recent findings suggest that tyrosylprotein sulfotransferases are involved in the regulation of tyrosyltransferase activity in proteins and peptides. These results indicate that tyrosine kinase 2 recognizes tyrosine residues adjacent to the acid residue for sulfation. This PTM leads to enhanced protein-protein interactions and is essential for secretory and transmembrane proteins. Some chemokine receptors, for example, HIV-1 and some In the N-terminal extracellular domain of CCR5, the major receptor for the glycoprotein hormone It has been shown that tyrosine sulfate is a sulfated thrombin inhibitor derived from leech. The natural form of the peptide hirudin is a sulfated tyrosine. Two recombinant forms of hirudin (Revasc and Refl) are used to treat coagulation disorders. udan) is not sulfated. Sulfation increases the mass of the biomolecule by 80 Da, which is The mass difference is the same as that of the phosphate moiety (PO3). PO forms a fairly stable PO bond. Unlike 3, SO3 is highly unstable and readily decomposes under high temperature and low pH conditions. .

[0003] The presence of different PTM variants in therapeutic antibody formulations may affect antibody potency, bioactivity, and efficacy depending on the location of the modification. This leads to heterogeneity that can result in variations in immunoavailability or immunogenicity. Such issues may also raise questions before regulatory agencies. Tyrosine sulfation is involved in the regulation of chemokine receptors. Although such modifications have been described in antibody preparations, it is unclear whether such modifications are possible in antibody preparations. We need to check whether they are present and, if so, remove them. Summary of the Invention

[0004] Overview of the Invention The present invention relates to anti-LAG3 antibodies or antigen-binding fragments thereof (e.g., Ab1, Ab 2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9). For example, CDR-L1 containing the amino acid sequence: KASQSLDYEGDSDMN (SEQ ID NO:38); CDR-L2 containing the amino acid sequence: GASNLES (SEQ ID NO:39); and CDR-L3 containing the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40) and / or a light chain variable domain comprising CDR-H1 containing the amino acid sequence: DYNVD (SEQ ID NO:33); Amino acid sequence: DINPNNGGTIYAQKFQE (SEQ ID NO:59); DINPNSGGTIYAQKFQE (SEQ ID NO:60); DINPNDGGTIYAQKFQE (SEQ ID NO:61); DINPNQGGTIYAQKFQE (SEQ ID NO:62); DINPNGGGTIYAQKFQE (SEQ ID NO:63); or DINPNX1GGTIYX2QKFX3X4 (SEQ ID NO: 64) (wherein X1=D, N , S or Q; X2=A or S; X3=Q or K; and X4=E or G). CDR-H2; and CDR-H3: NYRWFGAMDH (SEQ ID NO: 35); A heavy chain variable domain comprising which lacks detectable levels of sulfated tyrosine on CDR-L1. For example, In one embodiment of the invention, the antibody or fragment in the composition further comprises FR -L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4, FR-H 1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4 and a detectable level in one or more members selected from the group consisting of a constant domain. In one embodiment of the invention, the antibody or fragment lacks sulfated tyrosine. The components include engineered yeast or CHO N-linked glycans. In the present study, anti-LAG3 antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab 6, Ab7, Ab8 or Ab9)-containing compositions include one or more species of antibodies lacking tyrosine sulfation. and about 148590 Da, 148752 Da and / or 14891 Da. 4 Da molecular weight (e.g., as shown in Table 1, e.g., G0F and / or G1F groups) Lycan species, N-terminal heavy chain glutamine and / or lysine converted to pyroglutamate These include antibodies having the C-terminal heavy chain removed.

[0005] The present invention also provides antibodies that contain one or more sulfated tyrosines (e.g., on CDR-L1). or an antibody or antigen-binding fragment lacking an antigen-binding fragment and a sulfated tyrosine; and a tyrosine sulfated antibody or an antigen-binding fragment thereof (e.g., For example, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 ), the method comprising adjusting the pH of the mixture to about 6.5 to about 7.0, or to about 6. 5 to about 7.5, contacting the mixture with an anion exchange resin, and then removing the non-resin and removing and retaining the bound aqueous fraction from the resin. For example, the method involves washing a column with an aqueous composition under isocratic (isocratic) conditions. and removing and retaining the cleaning composition from the resin. In an embodiment, the resin is in a column and the method includes adding the mixture to the column; and and collecting the flow-through fraction from the column. The method includes the step of: The lipid was equilibrated in the chromatography column with 25 mM sodium phosphate (pH 6.5). The pH of the mixture is adjusted to about 6.5, the mixture is applied to a column, and the flow-through from the column is The fractions were collected and the resin in the column was washed with 25 mM sodium phosphate (pH 6.5). and collecting the flow-through fraction from the washing solution. In this case, the method comprises using a chromatograph containing a quaternized polyethyleneimine anion exchange ligand. The resin was packed in a chromatography column with 25 mM sodium phosphate (pH 7.0). (optionally 5 mM NaCl), adjust the pH of the mixture to about 7.0, and The column was then loaded with 25 mM riboflavin and the flow-through fraction was collected. sodium phosphate (pH 7.0) (optionally 5 mM NaCl) and then washed In one embodiment of the present invention, the method further comprises: collecting the flow-through fraction from the aniline solution. A of on-exchange chromatography flow-through 280 The absorbance is monitored, and A2 80 collect and retain the sample the first time it reaches at least about 2.5 absorbance units / cm; and A 280 Once it falls below about 1.0 absorbance units / cm, it is not collected or retained. In one embodiment of the present invention, the method further comprises the steps of: further anion exchange chromatography in bind-elute mode, hydrophobic interaction chromatography Chromatography, Protein A Chromatography, Protein L Chromatography, Protein G chromatography, hydroxyapatite chromatography, size exclusion Antibodies or In one embodiment of the present invention, the method includes purifying the antigen-binding fragment of the antibody or the antigen-binding fragment thereof. The immunoglobulin light chain and / or heavy chain of the antibody or antigen-binding fragment are In one embodiment of the invention, the gene is expressed in Indian hamster ovary cells. The antibody or antigen-binding fragment comprises: CDR-L1 containing the amino acid sequence: KASQSLDYEGDSDMN (SEQ ID NO:38); CDR-L2 containing the amino acid sequence: GASNLES (SEQ ID NO:39); and CDR-L3 containing the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40) and / or a light chain variable domain comprising CDR-H1 containing the amino acid sequence: DYNVD (SEQ ID NO:33); Amino acid sequence: DINPNNGGTIYAQKFQE (SEQ ID NO:59); DINPNSGGTIYAQKFQE (SEQ ID NO:60); DINPNDGGTIYAQKFQE (SEQ ID NO:61); DINPNQGGTIYAQKFQE (SEQ ID NO:62); DINPNGGGTIYAQKFQE (SEQ ID NO:63); or DINPNX1GGTIYX2QKFX3X4 (SEQ ID NO: 64) (wherein X1=D, N , S or Q; X2=A or S; X3=Q or K; and X4=E or G). CDR-H2; and CDR-H3: NYRWFGAMDH (SEQ ID NO: 35); The composition that is the product of such a method is also part of the invention. In one embodiment of the invention, anti-LAG3 antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab31, Ab32, Ab33, Ab34, Ab35, Ab46, Ab47, Ab58, Ab59, Ab60, Ab61, Ab62, Ab70, Ab81, Ab91, Ab92, Ab93, Ab94, Ab95, Ab106, Ab117, Ab123, Ab138, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab21, Ab22, Ab33, Ab b3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9) by AEX chromatography The antibody was purified by ELISA, where the antibody lacks sulfated tyrosines and has a molecular weight of approximately 148,590 Da, 14 8752 Da and / or 148914 Da (e.g., as shown in Table 1) As shown, for example, G0F and / or G1F glycan species, pyroglutamate, and a C-terminal heavy chain with glutamine and / or lysine removed. .

[0006] Detailed Description of the Invention Certain antibodies and other tags expressed in Chinese hamster ovary (CHO) cells Proteins are contaminated with sulfated tyrosine variants. Mass spectrometry of such variants reveals It is characterized by an adduct of about +80 Da, which corresponds to the mass of the added sulfate group. The adduct is also alkaline phosphatase resistant and reactive with anti-sulfated tyrosine antibodies. The present invention relates to a method for purifying compositions containing such contaminating tyrosine sulfate variants, as well as to a method for identifying such variants. An antibody composition that is substantially free of xenobiotics is provided.

[0007] The present invention relates to a method for the preparation of a medicament for use in the manufacture of a medicament for the preparation ... Recombinant DNA techniques may be used. Scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms include plurals and the plural forms Generally, the biochemistry, enzymology, molecular and and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization. Dimerization techniques and the nomenclature used therefor are well known in the art. Unless otherwise indicated, the method of the present invention The methods and techniques are generally described in various general terms and are cited and described throughout this specification. These are well known in the art and are described in more specific references. This is carried out according to conventional methods. For example, see James M. Cregg (ed.), Pichia Protocols(Methods in Molecular Biology) , Humana Press (2010), Sambrook et al., Molecular Cloning:A Laboratory Manual,2d ed.,Cold Spring Harbor Laboratory Press,Cold Spri ng Harbor, NY (1989); Ausubel et al., Current Pr. otocols in Molecular Biology,Greene Publ ishing Associates (1992 and supplements through 2002); rlow and Lane, Antibodies: A Laboratory Manu al,Cold Spring Harbor Laboratory Press,C Old Spring Harbor, NY (1990); Taylor and Dr. ickamer,Introduction to Glycobiology,Oxf ord Univ.Press(2003);Worthington Enzyme Manual,Worthington Biochemical Corp.,Fre ehold,NJ;Handbook of Biochemistry:Sect ion A Proteins,Vol I,CRC Press(1976);Han dbook of Biochemistry:Section A Proteins ,Vol II,CRC Press(1976);Essentials of Gl ycobiology,Cold Spring Harbor Laboratory Press(1999), Animal Cell Culture(RIFre) Immobilized Cells And Enz ymes(IRL Press,(1986));B.Perbal,A Practi See the Cal Guide To Molecular Cloning (1984). I want to.

[0008] Sulfated tyrosine includes tyrosine with a sulfate group added, for example having the following structure: [ka]

[0009] Chromatography The present invention relates to contaminating mutant antibodies or antigen-binding fragments thereof (e.g., For example, Ab1-Ab9) are combined with a composition (e.g., some of which have sulfated tyrosine and some is removed from a composition containing a mixture of antibodies or fragments lacking sulfated tyrosines , undetectable levels of tyrosine-sulfated variants (e.g., tyrosine-sulfated CDR-L1, The present invention provides a method for obtaining a composition comprising a β-amyloid β-amyloid (e.g., Ab1 or Ab6). In one embodiment, the composition is passed through a flow-through anion exchange (AEX) chromatography. The tyrosine sulfate variants are removed by chromatography. In the present study, the AEX resin is a dimethylaminopropyl ligand (i.e., dimethylaminopropyl). For example, in one embodiment of the invention, The composition to be subjected to AEX chromatography was purified by Protein A chromatography. In one embodiment of the present invention, AEX (e.g., dimethylaminopropane) is a product of Prior to treatment with Tris (e.g., 0.5M, 0 The composition is pH adjusted to a pH of about 6.5 with 0.725M or 1M. In terms of form, AEX columns (e.g., those with dimethylaminopropyl ligands) ) in, for example, 25 mM sodium phosphate, for example, phosphate buffer at pH 5, 6.2 or 6.5 In one embodiment of the present invention, the column (e.g., dimethyl sodium) is equilibrated. (having 25 mM dimethylaminopropyl ligand) in a buffer (e.g., 25 mM sodium phosphate The column is washed with sodium phosphate (e.g., sodium phosphate at pH 6.5) to remove the A It is possible to recover antibodies or fragments that are not tightly bound to the EX resin. Collect the flow-through that is not tightly bound to the AEX resin (e.g., in a fraction) and In one embodiment of the invention, after use, the column is, for example, Strip with 1M NaCl.

[0010] Mass spectrometry of the AEX flow-through material identified Ab6, which lacks tyrosine sulfation on CDR-L1. Several glycosylated species of the ribozymes are shown. These species are summarized in Table 1 below. The theoretical mass of is calculated by converting the N-terminal glutamine on the heavy chain to N-terminal pyroglutamic acid (pE1). and represents the calculated mass of an Ab6 molecule with the C-terminal lysine on the heavy chain removed (-K). . [Table 1]

[0011] The present invention relates to a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ... and having one or more molecular weights of about 148590, 148749, and / or 148915. and / or comprising the glycan species G0F and / or G1F, 3 antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, etc.) or Ab9; preferably Ab6).

[0012] The flow-through method is a method for recovering polypeptides by elution without using an elution step. In such a method, the term "purification of a polypeptide using a chromatography resin" is used. In this case, the polypeptide of interest does not bind tightly to the resin, but is removed from the polypeptide of interest. Contaminants that are detected bind tightly to the resin. For example, contaminant mutant antibodies with tyrosine sulfation A composition containing the antibody and an antibody lacking tyrosine sulfation is loaded onto a column containing AEX resin. and collecting and retaining the antibodies or fragments in the column flow-through. In a method comprising the steps of: (a) using an AEX resin in a flow-through format; The antibody is incubated under conditions that do not result in elution, e.g., isocratic conditions. In such a way, the contaminants can be washed away (and retained) from the column. The antibody that remains bound to the ram and lacks tyrosine sulfation remains in the flow-through. cormorant.

[0013] The binding / elution mode is a polysaccharide-binding assay using a chromatography resin by a method that includes an elution step. In such a method, the polypeptide of interest is bound to a resin. Contaminants that bind tightly to the resin but are removed from the polypeptide of interest In the case of a chromatography column, the contaminants flow through the column and are mostly bound to the resin. After optional washing, the bound antibody remains unbound from the resin. When exposed to an elution buffer that induces cleavage, they are dissociated, collected and retained.

[0014] Chromatographic resin ligands are used to bind desired molecules (e.g., Immobilized on stationary phase particles (e.g., Sepharose particles) that reversibly bind the antibody or contaminant It is a substance that has been

[0015] In one embodiment of the invention, the AEX resin is a ligand quaternized polyethyleneimine. (i.e., a ligand that includes a quaternized polyethyleneimine moiety). In an embodiment, a resin (e.g., one having a quaternized polyethyleneimine ligand) is In one embodiment of the invention, the resin (e.g., Those with quaternized polyethyleneimine ligands) are sodium phosphates (e.g., 25 The solution is equilibrated with NaCl (e.g., 5 mM) and NaCl (e.g., 5 mM) (pH about 7.0). In one embodiment, the column (e.g., having a quaternized polyethyleneimine ligand) The β-lactam ester is loaded with a feed medium, sodium phosphate (e.g., 25 mM) and The flow-through is washed with NaCl (e.g., 5 mM) (pH about 7.0), e.g., in fractions In another embodiment of the present invention, the present invention The disclosed method uses a chromatography resin containing an anion exchange ligand. The column is equilibrated with approximately 10 to 50 mM sodium phosphate at a pH of approximately 6.5 to 7.5, and mixed. The pH of the mixture is adjusted to about 6.5-7.5, the mixture is applied to a column, and the flow-through from the column is The column resin was washed with about 10 to 50 mM sodium phosphate buffer at a pH of about 6.5 to 7.5. The method includes washing the catalyst with thorium and collecting the flow-through fraction from the washing solution. In another embodiment, the method of the present invention comprises using a chromatographic column comprising an anion exchange ligand. The chromatographic resin is then mixed in a chromatography column at a pH of about 6.5 to 7.0 for about 10 to 50 ml. Equilibrate with 1 M sodium phosphate and adjust the pH of the mixture to approximately 6.5-7.0. Apply to the column, collect the flow-through fraction from the column, and infuse the resin in the column at a pH of approximately 6.5 to 7. Wash with approximately 10-50 mM sodium phosphate at 7.0°C, and then remove the flow-through from the wash. - collecting fractions.

[0016] Any suitable amount of the antibody or antigen-binding fragment may be applied to a chromatography resin, e.g. For example, chromatography columns (e.g., quaternized polyethyleneimine ligands or diphenyl It is possible to load AEX with ethylaminopropyl ligands. For example, in one embodiment of the present invention, a resin (e.g., a quaternized polyethyleneimine AEX with dimethylaminopropyl or dimethylaminopropyl ligands) approximately 10 per liter 0, 110, 120, 130, 140, 150, 100~150, 160, 170, 18 0, 190, 200, 300, 150-200, 100-200, 250-350 or Between 280 and 320 grams of material, e.g., an antibody or fragment, is loaded.

[0017] Chromatographic columns (e.g., quaternized polyethyleneimine ligands or dimethy When using an AEX resin with dimethylaminopropyl ligands, any Any acceptable dimensions may be used. For example, in one embodiment of the present invention, the column Diameter or height is approximately 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 cm.

[0018] The flow rate is determined by the amount of elution of the column (e.g., quaternized polyethyleneimine ligand or contains AEX resin with dimethylaminopropyl ligands) In one embodiment of the present invention, the flow rate is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 8 5, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210 and 215 liters per hour.

[0019] In one embodiment of the invention, a column (e.g., a quaternized polyethyleneimine ligase) is used. (containing AEX resin with dimethylaminopropyl or dimethylaminopropyl ligands) The absorbance of the through-flow liquid at 280 nm (A 280 ) is monitored. In one embodiment, the main A of the flow-through 280 Antibody or fragment in the peak In one embodiment of the present invention, the fragment products are collected and retained. 280 1c Approximately 1.0, 1.5, 2.0, 2.5 or 3.0 A per m (path length) 280 Absorbance Once the A is reached, collect the flow-through and 280 is approximately 1. A of 0, 1.5, 2.0, 2.5 or 3.0 280 Stop collection when absorbance falls below unit do.

[0020] Protect your chromatography columns from clogging due to particulate matter in the mobile phase (e.g., quaternization AEX resins with polyethyleneimine or dimethylaminopropyl ligands A pre-column filter can be used to protect the In one embodiment of the invention, the filter is a polyethersulfone membrane. Also, use a post-column filter to remove particulate matter from the flow-through. In one embodiment of the invention, the filter is 0.2 or 0.5 μm. The pore size is 2.0 mm.

[0021] The presence of variants with sulfated tyrosines can be determined, for example, by mass spectrometry of the flow-through fractions. The sulfated variants will have a higher mass than the non-sulfated variants. For example, In one embodiment of the invention, the sulfated variant is about 80% more sulfation-depleted than the variant lacking sulfation. In one embodiment of the present invention, sulfation is resistant to digestion by phosphatases. and sulfated peptides are more sensitive to electron transfer dissociation (E TD) have different fragmentation patterns.

[0022] In one embodiment of the invention, antibodies lacking tyrosine sulfation (e.g., Ab1, A b2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9, preferably Ab 6) A composition comprising a detectable tyrosine sulfation (e.g., in CDR-L1) It means a composition lacking detectable levels of tyrosine sulfation (e.g., CDR-L1 The composition includes a level not observable by mass spectrometry of the composition. For example, in one embodiment of the invention, mass spectrometry of the composition identifies the immunoglobulins of the composition. Mass measurements of intact and reduced forms of phosphopeptides and reduced peptide mapping were performed. In one embodiment of the present invention, reduced peptide mapping is performed using antibody immunoglobulins. Denaturation and reduction of purine disulfide bonds and alkylation of free cysteines, followed by This involves subsequent enzymatic digestion (eg, using LysC, trypsin or GluC). The enzyme digested peptides were analyzed by mass spectrometry. The "non-extractable" levels are less than about 0.5% (about 0.4, 0. 3, 0.2, or 0.1% or less of tyrosine sulfated species (e.g., on CDR-L1) Taste.

[0023] The molecular weight of a polypeptide can be determined, for example, by dividing the known weights of amino acids (modified or unmodified / sulfated). or non-sulfated) and known modifications (e.g., oxidation, deamidation, glycosylation, C and The molecular weight can be calculated based on the molecular weight of the target molecule (e.g., the N-terminal modification). In one embodiment of the present invention, the mass spectrometry can be performed by Quadrupole Time-of-Flight (Q-TOF) mass spectrometry or Orbitrap mass spectrometry.

[0024] The term "chromatography" refers to the contacting of a substance with a resin that acts as an adsorbent. A method for separating a solute of interest (e.g., a substance in a composition) from other substances in the composition by Adsorbents are sensitive to the properties of solutes, such as pI, hydrophobicity, size and structure. This allows the material to be more or less strongly adsorbed or retained under the specific buffer conditions of the method. Chromatography is the process of separating a composition through a bed of a chromatography resin, e.g., through a column containing the resin. Batch chromatographic purification can be carried out by conventional methods for permeation of the composition. and contacting the antibody or fragment-containing composition with the slurry to separate. The solution containing the substance that is not bound to the resin is The slurry is separated, for example, by allowing the slurry to settle and removing the supernatant, The unbound material can then be retained or discarded. Optionally, the slurry is subjected to one or more washing steps. Optionally, the slurry may be contacted with a suitable elution buffer to remove the resin-bound material. The material can be desorbed from the resin. The desorbed material can be retained or discarded. In one embodiment of the invention, the sulfated tyrosine variants of the antibody in the composition are anion-exchangeable. The tyrosine-sulfated mutants bound to the substituted resin whereas the non-sulfated tyrosine mutants did not bind significantly to the resin.

[0025] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is Purified by tein A or protein G chromatography. Protein G and Protein A is a serovar that binds to group G streptococci and Staphylococcus aureus (Stap It is a bacterial protein derived from Hylococcus aureus. The affinity of Protein G and Protein A for the Fc region is It forms the basis for the purification of IgG fragments containing IgG and IgG subclasses. Protein A or Protein G can be used for Protein A or Protein G chromatography. The present invention provides a method for detecting detectable levels of IgG, which can be bound to a solid phase such as sepharose. Production of compositions comprising antibodies or antigen-binding fragments thereof lacking tyrosine sulfate mutations Methods, or flow-through AEX chromatography and Protein A and and / or Protein G. or an antigen-binding fragment thereof.

[0026] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is It is purified by multimodal chromatography. Mixed-mode or mixed-mode protein chromatography is a method for detecting multi-modal interactions, e.g. ionic interactions. Exchange, hydroxyapatite, affinity, size exclusion and / or hydrophobic interactions The present invention is based on a resin functionalized with a ligand capable of detecting detectable levels of sulfated thiol. Methods for producing compositions comprising antibodies or antigen-binding fragments thereof lacking a rosin mutant, or flow-through AEX chromatography and mixed mode chromatography and removing sulfated tyrosine variants by a method comprising administering to said subject an antibody or an antigen-binding domain thereof. Methods for purifying the fragments are included.

[0027] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is Protein L is a chromatographic purification method for the purification of immunoglobulin light chains. Peptostreptococcus magnus binds to immunoglobulins via Reptococcus magnus protein. Protein L is a IgG, Binds to representatives of all antibody classes including IgM, IgA, IgE and IgD. Protein L is the variable domain of the kappa light chains of immunoglobulins and immunoglobulin fragments. Protein L binds to three of the four kappa light chain subtypes in humans ( 1, 3 and 4), which in mice bind to kappa 1. and a composition comprising an antibody or antigen-binding fragment thereof lacking a sulfated tyrosine variant of the polypeptide of claim 1. Production method or flow-through AEX chromatography and protein L chromatography and removing sulfated tyrosine variants by methods including chromatography using antibodies or A method for purifying the antigen-binding fragment is also included.

[0028] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is The purification is performed by hydrophobic interaction chromatography (HIC). The separation occurs when the proteins interact with the hydrophobic surface of the chromatography medium. The present invention is based on the reversible interaction between the tyrosine sulfate mutants and the tyrosine sulfate mutants. A method for producing a composition comprising an antibody or antigen-binding fragment thereof lacking the antibody or antigen-binding fragment thereof, or a flow cytometer Sulfated tyrosine mutations were detected by methods including AEX chromatography and HIC in a loop. The present invention also includes a method for purifying an antibody or antigen-binding fragment thereof to remove the antibody or antigen-binding fragment from a body fluid.

[0029] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is The product is purified by size exclusion chromatography (SEC), which separates the product based on differences in molecular size. The present invention isolates proteins that lack detectable levels of sulfated tyrosine variants. Method for producing a composition comprising an antibody or antigen-binding fragment thereof, or a flow-through Sulfuric acid was analyzed by methods including AEX chromatography and SEC chromatography. Method for purifying an antibody or antigen-binding fragment thereof to remove tyrosine-modified mutants - Patents.com Includes.

[0030] In one embodiment of the invention, the antibody or antigen-binding fragment is For example, in one embodiment of the present invention, the virus is inactivated. This is accomplished by pH treatment of a composition comprising an antibody or antigen-binding fragment thereof. Specifically, direct exposure of the composition to extreme pH can be used for viral clearance. For example, the pH treatment may be, in one embodiment of the present invention, a low pH treatment (e.g., pH 3 In one embodiment of the present invention, the antibody or antigen-binding fragment is In one embodiment of the present invention, the fragment is subjected to a high pH treatment. The neutralization may be achieved by removing a solvent or detergent from a composition comprising an antibody or antigen-binding fragment thereof. The present invention relates to antibodies or antibodies that lack detectable levels of sulfated tyrosine variants. The present invention relates to a method for producing a composition containing the antigen-binding fragment, or a method for producing a composition containing the antigen-binding fragment in a flow-through format. The method includes EX chromatography and viral inactivation to synthesize sulfated tyrosine mutants. The present invention also includes a method for purifying an antibody or antigen-binding fragment thereof to remove

[0031] "Ion exchange" separates molecules based on differences in their net surface charge. The charge characteristics of the ions are quite different, with their total charge, charge density and surface charge distribution being Depending on the difference, they exhibit different degrees of interaction with the charged chromatography resin. In one embodiment, the antibody or antigen-binding fragment thereof is chromatographed using ion exchange chromatography. "Ion exchange chromatography" is a method for purifying the cation exchange Chromatography, Anion Exchange Chromatography and Mixed Mode Chromatography Includes.

[0032] The term "ion exchange" resin refers to either negatively charged (i.e., cation exchange) or positively charged By "electrochemically charged" is meant an anion-exchange solid phase.

[0033] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is The product is purified by cation exchange chromatography. A "cation exchange" resin is a resin that carries a negatively charged a solid phase having free cations for exchange with cations in an aqueous solution passing through the solid phase; Any solid phase bonded to a suitable solid phase to form a cation exchange resin. Negatively charged ligands can be used. For cation exchange materials, the ligand: sulfopropyl (SP ) -CH2-CH2-CH2-SO3 - ;Methylsulfonate (S) -CH2-SO 3 - ; or carboxymethyl (CM) -CH2-COO - Although some have However, the present invention is not limited to these. Methods for preparing compositions comprising antibodies or antigen-binding fragments thereof that lack a target polypeptide Includes through-AEX and cation exchange chromatography The present invention relates to a method for removing sulfated tyrosine variants by the use of an antibody or antigen-binding fragment thereof. This includes a method for purifying the compound.

[0034] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is The product is purified by anion exchange chromatography. "Anion exchange" resins are resins that carry positively charged It means a solid phase having one or more positively charged ligands attached thereto. Any positively charged ligand that is attached to a solid phase suitable for forming an ion exchange resin can be used. The anion exchange material has the ligand: quaternary ammonium (Q) -CH2-N + -(CH3 )3-;Diethylaminoethyl (DEAE) -CH2-CH2-N + -(CH2-CH 3)2; or diethylaminopropyl (ANX) -CH2-CHOH-CH2-N + These include, but are not limited to, those having -(CH2-CH3)2. The GoPure D 50 μm column has a dimethylaminopropyl functional group. and an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine mutants. A method for producing a composition containing the compound, or a method for producing a composition containing the compound by AEX chromatography in a flow-through mode and AEX chromatography (bind / elute mode) to identify sulfated tyrosine variants. The present invention also includes a method for purifying an antibody or antigen-binding fragment thereof to remove

[0035] The term "solid phase" or "stationary phase" refers to a solid phase that is a mixture of one or more ligands (e.g., anion exchange ligands). "Aqueous matrix" refers to any non-aqueous matrix to which a ligand (such as an ion exchange ligand or a cation exchange ligand) can be attached. or, in the case of size exclusion chromatography, it is used as a gel of the resin. The mobile phase can refer to a liquid that is separated from the solid phase, e.g. in a chromatographic purification. The mobile phase is an aqueous substance that carries the antibody or antigen-binding fragment on the column. A loading buffer may be included to be applied. A loading buffer may be used to form a solid phase. Examples of materials include polysaccharides (e.g., agarose and cellulose) and other mechanically stable Matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene These include benzene, polyacrylamide, ceramic particles and derivatives of any of these. do.

[0036] Loading a mixture containing antibodies or antigen-binding fragments for purification Before the run, use an "equilibration" buffer to adjust the pH and conductivity of the chromatography resin. Suitable buffers or solutions that can be used for this purpose are The buffers are well known in the art (e.g., those described above) and are suitable for purifying a protein of interest. at a pH compatible with the selected resin used in the chromatographic step for This includes any buffer of

[0037] The mixture containing the antibody or antigen-binding fragment is immersed in a purification resin (e.g., an anion exchange resin). A "loading" buffer is used to load the resin onto the cation exchange resin (or cation exchange resin). Any suitable solution may be used as the loading buffer. In one embodiment of the present invention, the loading buffer is an elution buffer. It is prepared from a buffered mixture resulting from a previous purification step such as

[0038] The term "wash" buffer or solution refers to a solution that is used to elute the antibody or antigen-binding fragment. Before the purification resin (e.g., anion exchange resin or cation exchange resin) is The term "washing" refers to a composition that is used to dissolve chromatographic materials. In one embodiment of the present invention, the cleaning solution is Isocratic (uniform concentration). Chromatography under isocratic wash conditions. The mobile phase remains essentially the same.

[0039] Tyrosine sulfate mutant antibodies and antigen-binding fragments are contaminants ), but the present invention also relates to, for example, The present invention also includes compositions comprising such variants that are not bound in the absence of non-tyrosine sulfated variants. Non-binding variants are AEXs after removal from non-tyrosine sulfated antibodies and fragments. It can be obtained by elution from the column.

[0040] The "elution" buffer is used to elute the molecules (e.g., antibodies or The antigen-binding fragment thereof is then dissociated.

[0041] Upstream Processing Antibodies and antigen-binding fragments purified from contaminating tyrosine sulfate mutants are For example, the method of the present invention can be produced by cell expression. Prior to removal of the foreign bodies, conditions favoring such expression of the antibody or antigen-binding fragment are established. Expression of heavy and / or light immunoglobulin chains in a host cell in a medium under The present invention includes the isolation of detectable levels of sulfur from host cells and / or culture media. Methods for Producing Compositions Comprising Antibodies or Antigen-Binding Fragments Lacking Oxidized Tyrosine Mutations - Patent application or host cell expression and flow-through AEX chromatography. or antigen-binding fragment thereof for removing sulfated tyrosine mutants by a method according to the present invention The present invention includes a method for purifying the above-mentioned compound.

[0042] The scope of the present invention includes antibodies that do not contain tyrosine sulfation (e.g., on CDR-L1). or an antigen-binding fragment thereof, the method comprising: (i) administering to said patient a composition comprising said antigen-binding fragment; Polynucleotides encoding the immunoglobulin light and / or heavy chains of the antibody or fragment (ii) introducing the leucine into a host cell (e.g., a CHO cell); and (iii) inducing the immune response in the cell. The host cells are cultured under conditions favoring expression of the immunoglobulin chains, e.g., The antibody or antigen-binding fragment having the phosphoryl chain is secreted from the host cell into the culture medium and and (iii) using the anion exchange chromatography in a flow-through mode as described herein. and extracting immunoglobulin chain polypeptides from host cells and / or medium by methods including filtration. This involves isolating the

[0043] For example, the antibody or fragment may be subjected to cell lysis, e.g., by grinding / scraping (e.g., by grinding with a glass beads), French press cell lysis, enzymatic digestion or sonication The soluble and insoluble materials obtained by the method can be released from the host cell. The lysed cells containing the sulfated tyrosine mutants constitute a cell lysate. Methods for producing compositions containing antibodies or antigen-binding fragments thereof lacking the and flow-through AEX chromatography to detect sulfated tyrosine. Methods for purifying the antibody or antigen-binding fragment thereof to remove foreign bodies are included.

[0044] In one embodiment of the invention, the antibody or antigen-binding fragment is purified by centrifugation. Centrifugation of the cell lysate or other suspension may be used to obtain the antibody or This removes most particulate matter, such as cell debris, from the aqueous fraction containing the fragments. For example, in one embodiment of the invention, centrifugation is performed by centrifuging the lysate (e.g., Approximately 40,000-50,000 for cell lysate (including discarding the lysate solid fraction) In one embodiment of the present invention, centrifugation is performed at 10,000×g for 15 to 30 minutes. For example, in the range of about 8,000 x g to about 15,000 x g, the cells are removed from the liquid medium. Gravity within the range (e.g., about 8000, 9000, 10000, 11000, 12000, 1 3000, 14000 or 15000) (e.g., about 0.9 × 10 -8 ~2.8×10 9 The centrifugation is performed using a method characterized by a Q / SIGMA ratio in the range of 0.01 to 0.01. In one embodiment, the liquid centrifuged solution is filtered through a depth filter (e.g., 0.1 to about 0 The present invention relates to antibodies lacking sulfated tyrosine mutants or The present invention relates to a method for producing a composition comprising the antigen-binding fragment, or a method for centrifugation and flow cytometry. The removal of sulfated tyrosine variants by a method involving through-AEX chromatography The present invention includes a method for purifying an antibody or an antigen-binding fragment thereof for the purpose of detecting an antibody or an antigen-binding fragment thereof.

[0045] In one embodiment of the invention, the immunoglobulin heavy and light chains are It is expressed in a host cell fused to the sequence and secreted from the host cell into the host cell medium. .

[0046] In one embodiment of the invention, the antibody or antigen-binding fragment is filtrated (e.g. For example, the present invention is purified by AEX chromatography (e.g., before or after AEX chromatography purification). In one embodiment, the aqueous composition comprising the antibody or antigen-binding fragment comprises: For example, through a filter having a pore size of about 1 μm, 0.45 μm, or 0.22 μm. In one embodiment of the present invention, the filtration is performed to remove solid particulate matter. The filters are made from cellulose acetate or polyvinylidene fluoride (PVDF). The present invention relates to compositions comprising antibodies or antigen-binding fragments thereof that lack sulfated tyrosine mutations. The process involves the manufacture of the composition or flow-through AEX chromatography and filtration. and removing tyrosine sulfate variants by a method comprising administering to said subject an antibody or an antigen-binding fragment thereof. This includes a method for purifying the ment.

[0047] In one embodiment of the invention, the antibody or antigen-binding fragment is subjected to fractionation precipitation. Increasing the salt concentration enhances the hydrophobic interactions between proteins, leading to selective precipitation. In one embodiment of the invention, an aqueous solution containing the antibody or fragment is The composition comprises ammonium sulfate, dextran sulfate, polyvinylpyrrolidine, polyethylene glycol (PEG; e.g., PEG4000), acetone, polyethyleneimine, sulfuric acid The precipitation is carried out in the presence of protamine, streptomycin sulfate or caprylic acid. and preparing a composition comprising an antibody or antigen-binding fragment thereof lacking a sulfated tyrosine mutation. or flow-through AEX chromatography and fractional precipitation. The present invention relates to a method for removing sulfated tyrosine variants by the method of the present invention. This includes a method for purifying the compound.

[0048] In one embodiment of the invention, the host cell in which the immunoglobulin chains are expressed is a mammalian Cells such as Chinese hamster ovary (CHO) cells, mouse myeloma cells, PER A cell, a hybridoma cell, or a fungal or yeast cell, such as a Pichia ), such as Pichia pastoris or Saccharomyces cerevisiae. The present invention is directed to Saccharomyces cerevisiae. In one embodiment, the host cell, e.g., a CHO cell, expresses glutamine synthase. Lacking.

[0049] In one embodiment of the invention, a nucleic acid encoding an immunoglobulin heavy and / or light chain is The polynucleotide is operably linked to one or more expression control sequences, e.g., a promoter. For example, immunoglobulins are present in expression vectors. To achieve expression of the antigen-binding fragment, a strong promoter / enhancer is used. , e.g., the cytomegalovirus (CMV) promoter and / or elongation factor alpha The (EF1α) promoter is used to drive the expression of immunoglobulin heavy and / or light chains. It is possible to drive it.

[0050] In one embodiment of the present invention, a method for enhancing the export of transcribed mRNA from the nucleus to the cytoplasm is provided. To achieve this, intron sequences in the 5' untranslated region are included after the promoter / enhancer. Rarely, they contain one or more 3' polyadenylation signal sequences to maximize mRNA levels. In one embodiment of the invention, the polyadenylation signal sequence is Late or early polyadenylation signal sequence or bovine growth hormone polyadenylation sequence In one embodiment of the present invention, the first translation initiation sequence is By placing GCC GCC(A / G)CC (SEQ ID NO:69) immediately before the start codon In one embodiment of the invention, an antibody or A signal peptide sequence immediately preceding the immunoglobulin chain to direct secretion of the immunoglobulin or fragment. will be placed.

[0051] Cell culture conditions can be monitored and adjusted as necessary. For example, pH, cell culture temperature, and cell density. Conditions such as cell number, cell viability and temperature can be monitored and adjusted. In an embodiment, the temperature of the cell culture is, for example, 37°C to 30°C at 48 hours after inoculation. The temperature is adjusted to 35° C. In one embodiment of the present invention, dissolved oxygen is monitored and and / or adjusted to a set value such as 20-50%. In this case, dissolved CO2 is monitored and / or maintained at a constant level, e.g., above about 120-150 mmHg. In one embodiment of the invention, the osmolality is monitored and / or Or, for example, it is adjusted to about 270 to 330 mOsm / kg.

[0052] antibody The present invention provides an antibody and its antigen-binding fragment that lacks detectable levels of sulfated tyrosine. Compositions containing antibodies and fragments thereof, and methods for isolating compositions containing such antibodies and fragments For example, in one embodiment of the invention, the antibody or fragment is Contains sulfated tyrosine, PD1, CD27, LAG3, CTLA4, BTLA, TIM3 , ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1, PD-L2 , ILT1, ILT2, CEACAM1, CEACAM5, TIM3, TIGIT, VI STA, ILT3, ILT4, ILT5, ILT6, ILT7, ILT8, CD40, O X40, CD137, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL 4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3 DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17 or TSLP The antibody binds to an antigen selected from the group consisting of

[0053] The "LAG" polypeptides to which the antibodies and antigen-binding fragments of the present invention bind The term "3" refers to the human and cynomolgus monkey strains, e.g., Macaca fascicularis (Macaca ca fascicularis) or Macaca mulatta ta) LAG3, as well as fragments thereof, e.g., the mature fragment thereof lacking the signal peptide. Taste.

[0054] Anti-LAG3 antibodies lacking tyrosine sulfation (e.g., those disclosed in WO2016028672) Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 Examples of immunoglobulin chains of the IgG family include those summarized below. For example, The antibody or fragment may comprise any of the CDRs and / or immunoglobulins described below. In one embodiment of the invention, the contaminating antibody or antigen-binding fragment comprises one or more of the The fragment is the amino acid sequence KASQSLD Y EGDSDMN (SEQ ID NO: 38) Contains CDR-L1, where Y (underlined and bold) is sulfated.

[0055] In one embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment is , 4A10 heavy and / or light chain immunoglobulins; V H and / or is V L or light chain CDRs and / or heavy chain CDRs (e.g., CDR- L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) Includes.

[0056] In one embodiment of the invention, Ab1, Ab2, Ab3, Ab4, Ab5, Ab 6, Ab7, Ab8 or Ab9, any N-terminal heavy chain glutamine was a peak. is converted to 2-aminoglutamate and / or any C-terminal heavy chain lysines are removed. [ka]

[0057] In one embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment is 19E8 heavy and / or light chain immunoglobulins; V H and / or V L or light chain CDRs and / or heavy chain CDRs (e.g., CDR-L of 19E8 1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) include. [ka]

[0058] In one embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment is 11C9 heavy and / or light chain immunoglobulins; V H and / or V L or light chain CDRs and / or heavy chain CDRs (e.g., CDR-L of 11C9 1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) include. [ka] TIFF0007604436000006.tif21152

[0059] In one embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment is 22D2 heavy and / or light chain immunoglobulins; V H and / or V L or light chain CDRs and / or heavy chain CDRs (e.g., CDR-L of 22D2 1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) include. [ka] TIFF0007604436000008.tif49152

[0060] In one embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment is Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 heavy chain Immunoglobulins and / or light chain immunoglobulins; V H and / or V L Chains, or is a light chain CDR and / or a heavy chain CDR (e.g., Ab1, Ab2, Ab3, Ab4, CDR-L1, CDR-L2, CDR-L of Ab5, Ab6, Ab7, Ab8 or Ab9 3, CDR-H1, CDR-H2 and CDR-H3). [ka] TIFF0007604436000010.tif232153TIFF0007604436000011.tif226152

[0061] TIFF0007604436000012.tif226153TIFF0007604436000013.tif235155TIFF0007604436000014.tif129148

[0062] TIFF0007604436000015.tif90147TIFF0007604436000016.tif224153TIFF0007604436000017.tif231154TIFF0007604436000018.tif76152

[0063] In one embodiment of the invention, any anti-LAG3 antibody or its antigen binding molecule of the invention is The CDR-H2 of the synthetic fragment has the amino acid sequence: DINPNX1GGTIYX2QK FX3X4 (SEQ ID NO: 64), wherein X1=D, N, S or Q; X2=A or S; X3=Q or K; X4=E or G.

[0064] The present invention relates to the use of N-linked glycans in Chinese hamster ovary cells (CHO) or engineered engineered yeast cells (engineered yeast N-linked glycans), e.g., Pichia pastoris N-linked glycosylation typically added to immunoglobulins produced in Bacillus pastoris Antibodies and antigen-binding fragments thereof, including cans (e.g., 4A10, 19E8, 11 C9 and / or 22D2; e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab For example, in one embodiment of the present invention, In this case, the antibody or antigen-binding fragment is an "engineered yeast N-binding domain" shown in FIG. "conjugated glycans" or "CHO ​​N-linked glycans" (e.g., G0 and / or G0-F and / or G1 and / or G1-F and / or G2-F and / or Ma n5). In one embodiment of the invention, the antibody or antigen-binding fragment The segments are engineered yeast N-linked glycans, i.e., G0 and / or G1 and / or comprises G2 and optionally further comprises Man5. In one embodiment of the invention, The antibody or antigen-binding fragment may comprise CHO N-linked glycans, i.e., G0-F, G1-F and G2-F, and optionally further comprising G0 and / or G1 and / or In one embodiment of the invention, the antibody or Approximately 80% to approximately 95% of all N-linked glycans on the immunoglobulin chains of the antigen-binding fragment (e.g., about 80-90%, about 85%, about 90%, or about 95%) of the engineered yeast N-linked glycoproteins Nett et al., Yeast. 28(3):23 7-252(2011);Hamilton et al., Science.313(5792): 1441-1443(2006); Hamilton et al., Curr Opin Biot See, e.g., Echnol. 18(5):387-392 (2007). In one embodiment, the engineered yeast cell is GFI5.0 or YGLY8316. or U.S. Pat. No. 7,795,002 or Zha et al., Methods Mol The strain is described in International Patent Application Publication No. See also publication number WO2013 / 066765.

[0065] Anti-LAG3 antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7 , Ab8 and / or Ab9) are tyrosine sulfated variants of approximately 148670 Da, 14 Contains a molecular weight of 8832 Da and / or 148994 Da. Variants have molecular weights of approximately 148590 Da, 148752 Da and / or 148914 Da. Includes.

[0066] An "isolated" antibody or antigen-binding fragment thereof is one that is isolated from the cell in which it is produced or from which it is isolated. or other biological molecules from cell culture. Biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell debris and growth media. The isolated antibody or antigen-binding fragment may further be administered to a host cell. At least in part, the expression system components, such as biological molecules derived from the cells or from their growth medium, In general, the term "isolated" refers to such a biological fraction. The complete absence of the antibody or the fragment, or the absence of water, buffer or salt, or the presence of the antibody or the fragment. It does not refer to a component of a pharmaceutical preparation that contains a fragment.

[0067] An antigen-binding fragment of an antibody specifically binds to the antigen bound by the full-length antibody. Examples of antigen-binding fragments include Fab, Fa, and Fab fragments. b', F(ab')2 and Fv fragments; diabodies; single chain antibody molecules, such as s cFv; and nanobodies and multispecific antibodies formed from antibody fragments. These include, but are not limited to:

[0068] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer is made up of two polypeptide chains. Each pair has one "light" and one "heavy" chain. , Fundamental Immunology Ch.7 (edited by Paul, W., 2n d ed. Raven Press, NY (1989).

[0069] Monoclonal antibodies are substantially homogeneous antibodies, i.e., the antibody fraction that makes up the population is The offspring are identical in amino acid sequence, except for possible natural mutations that may exist in small amounts. Kohler et al. (1975) Nature 256:495; U.S. Patent No. 4 ,816,567;Clackson et al.,(1991)Nature 352:624 -628; Marks et al. (1991) J. Mol. Biol. 222:581-597 ; and Presta (2005) J. Allergy Clin. Immunol. 1 See 16:731.

[0070] A chimeric antibody has the variable domains from a first antibody and the constant domains from a second antibody. antibodies, where the first and second antibodies are from different species (see U.S. Pat. No. 6,393,623). No. 4,816,567 and Morrison et al. (1984) Proc. Natl. A cad. Sci. USA 81:6851-6855). Typically, the variable domain is The antibody is derived from an antibody from a test animal (the "parent antibody"), e.g., a rodent, and the constant domain sequences are human. The resulting chimeric antibodies are likely to elicit adverse immune responses in human subjects. This will be less likely to occur than with the parent (eg, murine) antibody.

[0071] Humanized antibodies are derived from both human and non-human (e.g., mouse or rat) antibodies. Generally, a humanized antibody contains at least one, and typically two, variable domains. and substantially all of the hypervariable loops are non-human immunoglobulins. corresponding to those of globulins and all or substantially all of the framework (FR) regions All are of human immunoglobulin sequences. Humanized antibodies can optionally contain human immunoglobulins. It may comprise at least a portion of the Brin constant region (Fc).

[0072] Immunoglobulins are classified into different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are at least five major classes of immunoglobulins: IgA, IgB, IgC, IgD, IgE ... There are three types of IgA antibodies: IgD, IgE, IgG, and IgM, some of which are further divided into subclasses ( isotype), e.g., IgG-1, IgG-2, IgG-3 and IgG-4; IgA The present invention relates to antibodies of these classes or subclasses. The present invention includes any antibody or antigen-binding fragment thereof (e.g., anti-LAG3).

[0073] In one embodiment, an antibody or antigen-binding fragment (e.g., anti-LAG3 ) is a heavy chain constant region, e.g., a human constant region, e.g., γ1, γ2, γ3 or γ4 human heavy chain constant region. In another embodiment, the antibody or antibody fragment comprises an antibody chain constant region or a variant thereof. The original binding fragment (e.g., anti-LAG3) may comprise a light chain constant region, e.g., a human light chain constant region. Human light chain regions, such as lambda or kappa human light chain regions or variants thereof. Although not essential, the human heavy chain constant region can be γ4, and The light chain constant region can be kappa. In another embodiment, the antibody The Fc region of is γ4 with a Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38:1-8, 2001). [Brief description of the drawings]

[0074] [Figure 1] Overlay of IEX-HPLC UV profiles of the AEX feed (bold trace), strip (thin trace) and pooled fractions (dashed trace). [Diagram 2] Intact mass spectra of AEX feed, pool and strip samples. [Diagram 3] Reduced light chain mass spectra of AEX pool and strip samples. [Figure 4] Figure 4A-B: UV traces of reduced LysC peptide mapping of the AEX pool and stripped fractions. [Diagram 5] Figure 5A-C. CID fragmentation spectra of light chain AA25-43 + 80 Da at (A) 400-1800 m / z, (B) expanded from m / z 300-1100, and (C) expanded from m / z 1200-2000. [Figure 6] ETD fragmentation of light chain peptide AA25-43 + 80 Da. The 80 Da bond fragment ion was labeled. [Figure 7] 7A-B. (A) Deconvoluted intact mass spectrum of AEX strip fractions with and without alkaline phosphatase treatment. (B) Deconvoluted intact mass spectrum of chicken ovalbumin with and without alkaline phosphatase treatment. [Figure 8]Figure 8A-B. (A) Normalized concentrations of mAb AEX pools and strips were subjected to reducing SDS-PAGE and probed for human heavy chain (HC) and light chain (LC) by Western hybridization (top panel), then stripped and reprobed for anti-sulfotyrosine (bottom panel). See HC and LC designations on the far right. (B) In addition to AEX strips and pools, normalized concentrations of different CHO-derived mAbs were subjected to reducing SDS PAGE and probed for human HC and LC by Western hybridization, then stripped and reprobed for anti-sulfotyrosine. For both (A) and (B), MagicMark XP was used as a protein molecular weight standard. Equal amounts of HEK293 and EGF-treated A431 cell extracts are analyzed as controls. [Figure 9] Figure 9A-C. SIC of (A) LC25-43 + 80Da from AEX strip fraction, (B) synthetic peptide XSXSXDYEGDSDXXXXXXX (SEQ ID NO: 65) + phosphorylated, and (C) synthetic peptide XSXSXDYEGDSDXXXXXXX (SEQ ID NO: 65) + sulfated. [Figure 10] mAb tyrosine (Y31) site showing the CDR loops in ribbon diagrams for both the heavy and light chains. [Figure 11] Major N-linked glycans for monoclonal antibodies produced in Chinese hamster ovary cells (CHO N-linked glycans) and for monoclonal antibodies produced in engineered yeast cells (engineered yeast N-linked glycans); squares: N-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

[0075] Working Example These examples are illustrative of the invention and are not intended to be limiting thereof.

[0076] Example 1: A method for identifying and purifying antibody tyrosine sulfate mutants.

[0077] In this example, the presence of tyrosine sulfated antibody variants of Ab6 was identified and the variants were A purification method was developed to remove

[0078] material and method Alkaline phosphatase was obtained from New England Biolabs (Ipswich, Anti-tyrosine sulfate antibodies were obtained from Millipore (Billie, MA). Synthetic peptides were obtained from AnaSpec (Fremont, TX). nt, CA).

[0079] Anion exchange (AEX) chromatography By using the GE Akta Avant system, POROS GoPure D A prepacked column (0.5 × 5 cm, 1 mL) was used in the flow-through mode to measure the AEX clathrate. Protein A chromatography-purified mAb was diluted with 1M Tri The pH was adjusted to 6.5 with 500 mL of 10 ... Prior to loading, the column was equilibrated with 25 mM sodium phosphate (pH 6.5). The column was then washed with 25 mM sodium phosphate (pH 6.5) and stripped with 1 M NaCl. The absorbance at 280 nm was monitored during the run. The AEX loads were collected and analyzed.

[0080] Ion Exchange HPLC An Agilent 1600 series system was used, and a MabPac SCX-10 column ( Ion-exchange HPLC was performed at ambient temperature on a column (4 x 250 mm, 3.14 ml). Mobile phase B The mobile phase A was 30 mM sodium phosphate (pH 8.0), and the mobile phase B was 25 mM MES (pH The column was first run at a flow rate of 1.0 mL / min with 14% mobile phase B for 10 min. The mAb tandem was then equilibrated using a gradient of mobile phase B (14% to 80% in 18 min). The protein was eluted from the column. The column was then washed with 100% mobile phase B for 3 min, and the next sample was The column was re-equilibrated with 14% mobile phase B for sample analysis. The absorbance of the eluate at 280 nm was measured. C was monitored throughout the implementation.

[0081] Intact and reduced LC / MS 20 μg of sample was diluted to 0.5 mg / m in 50 mM Tris buffer (pH 8.0). The mixture was diluted to 1 L using a Waters Acquity UPLC H-Class. RP-HPLC separation was performed using an Acquity UPLC BEH30 column. 0 C4,1.7μm,1.0×100mm(Waters,Milford,MA;- The mobile phase was: Mobile phase A: 0.1% formic acid (FA) in water and 0.1% in acetonitrile (ACN) as mobile phase B. The LC flow rate was 0.08 mL / min, and the column temperature was maintained at 80°C. The antibodies were eluted using a 4-15 min gradient of 30% to 90% B. A Waters Xevo G2 Q-TOF system with a scanning range of 4000 was used. I got the spectrum.

[0082] 20 μg of sample was diluted in reducing buffer (50 mM Tris (pH 8.0) to a final volume of 100 μL. M Dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, M0 solution was added to each of the samples, followed by incubation at 56° C. for 20 minutes. RP-UPLC separation was performed on a Waters Acquity UPLC H-Class. The column used was Acquity UPLC, BEH300 C4, 2.1 x 100 m. m, 1.7 μm (Waters). Scanning range m / z 600-3000. MS spectra were obtained on a Waters Xevo G2 Q-TOF system. was analyzed by MaxEnt1 in MassLynx 4.1.

[0083] Peptide Mapping LC / MS 100 μg of sample was diluted with 50 mM Tris (pH 8.0), 6 M guanidine HC1 The buffer was exchanged with 100 μL of denaturation buffer containing 1 mM EDTA and 5 mM The reduction reaction was carried out at 56 °C for 30 min using 20 mM DTT in the solution. The resulting mixture was alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark. The reaction was terminated by adding 1 μL of 500 mM DTT solution. Dilute the alkylated sample in 300 μL of digestion buffer (50 mM Tris pH 8.0). Lys-C enzyme (Wako, Richmond, VA) was diluted to a final volume of 1: The enzyme:substrate ratio was 20 (w:w). The solution was incubated at 37°C for 4 hours. ALO Peptide ES-C18, 2.1×150nm, 2.7μm column (MA C-MOD Analytical, Inc., Chadds Ford, PA) was used. Then, the RP-HPLC was performed using a Waters Acquity UPLC H-Class. Peptides were separated using a Waters HPLC system scanning in the m / z range of 100-2000. MS spectra were acquired on a Xevo G2 Q-TOF system. MS data were submitted to Biopharmaceutical Analysis was performed using rmaLynx 1.3 (Waters).

[0084] Targeted MS / MS LC / MS / MS of the target peptides was performed using an LTQ-Orbitrap MS system (Therm o Fisher, Waltham, MA). 17500 resolutions in FT mode. The HALO Peptide ES-C18 capillary was used for MS / MS acquisition. A Waters Acquity microscope was used to image the specimen. Peptides were separated by UPLC H-Class. The MS / MS was scanned over the m / z range. The normalized fragmentation energies were calculated as 35% for CID fragmentation and 35% for ETD fragmentation was set to 35%. MS2 data were interpreted manually.

[0085] Alkaline phosphatase treatment 10 μg of mAb protein in the AEX strip fraction was diluted with 50 μL of phosphatase Dilute in reaction buffer. 1 μL (10 units) of alkaline phosphatase from calf intestine Add 100 mL of lyase (New England Biolabs, Ipswich, MA) and mix for 3 h. The mixture was incubated at 7°C for 1 hour. Chicken ovalbumin (Sigma) was used as a positive control. 10 μL of the solution was injected into the LC / MS for mass spectrometry.

[0086] Western blotting Magic Mark XP Western Standard (Invitrog en) and specific concentrations of monoclonal antibodies (mAbs) and control cell extracts (HEK 293 whole cell extracts and EGF-stimulated A431 cell lysates (Millilpore) ) were reduced with β-mercaptoethanol with heating at 95°C, and then A 4-20% gradient gel (Novex) was used to measure the chromatograms based on Tris-glycine. The proteins were separated by SDS PAGE. The separated proteins were then transferred to a nitrocellulose membrane. The plate was electrophoretically transferred to a 100-well plate and incubated at 4°C with rocking in Tris-buffered saline + 0.05% Tween 100 ml. The membrane was then washed overnight in Tris-buffered saline (TBST) (Sigma). + 1% BSA (TBS-BSA) (Sigma) at room temperature with continuous rocking. Blocking was performed for 1 hour. Primary antibody (anti-sulfotyrosine / anti-tyrosine sulfate (Milli pore) or anti-human IgG (H+L) (Jackson ImmunoResearch ch Laboratories Inc.) in TBS-BSA, and The membrane was incubated at room temperature for 2 hours. After washing with TBST, HRP-conjugated secondary antibody was added. 5% goat anti-mouse or goat anti-rabbit (Thermo Scientific) Skim milk protein + 0.05% Tween 20-phosphate buffered saline (Invi The samples were diluted in TBST and incubated at room temperature for 1 hour. After final washing, a chemiluminescent substrate (Thermo Scientific) was used for development. Signals captured on photographic film (GE Healthcare Life Sciences) The nitrocellulose membrane was stripped between primary antibodies and subsequently processed. Stripping was performed as previously described (Kaufmann SH, EC, Shaper JH.,The erasable Western blot.Ana l Biochem., 1987.161(1):p.89-95).

[0087] Results and Discussion Isolation of mAb molecules Anion exchange chromatography (AEX) is typically used in monoclonal antibody purification. This step is typically used as a polishing step in a flow-through The method is operated in a mode where the mAb flows through the column and impurities (HCPs, D During the development of AEX, the column was loaded with NA and bound to the resin. It was found that some of the mAbs used affected protein recovery. The fraction was eluted in the strip fraction of the AEX chromatography. To characterize the mAb, fractions from the AEX chromatography were analyzed: "load ( "Loading" refers to the sample before AEX purification; "Pool" refers to the sample and the "strip" refers to the bound fraction of the sample. Loading, Pooling and Stripping from AEX Chromatography The fractions were first analyzed by IEX-HPLC chromatography. IEX-HPLC profiles of mAb in pooled, pooled and stripped fractions are shown. As shown in Figure 1, the strip fraction showed significantly higher ELISA counts compared to the load and pool fractions. It contains a high amount of acidic variants: 2 in the pooled fraction (dashed trace) 3% in the stripping fraction, compared to 33% in the feed fraction (bold trace). In the top fraction (thin trace), it is ~65% acid variant. Additional differences were observed in the AEX feed. This peak was observed at higher levels in the AEX feed. In the AEX pool, this peak was minimal. The fractions were enriched in the acidic pre-major peak. AEX chromatography was performed at pH 7.0 or 7.5. A similar decrease in the acidic pre-major peak was observed in the AEX pool.

[0088] Analysis of intact and reduced proteins by mass spectrometry LC / MS of intact and reduced forms with Q-TOF MS to characterize impurities All three fractions (AEX load, pool and strip) were analyzed by S. shows the deconvoluted mass spectrum of the intact molecule. The three major glycoforms, masses of 148591 Da, 148751 Da and 148912 Da, respectively. G0F / G0F, G0F / G1F and G1F / G1F were detected in all three fractions. The calculated mass of the intact form of this molecule with G0F / G0F is 148590 The mass measurements of the intact form are all within 25 ppm of mass error. The additional species is A These species were only detected in the EX strip fraction. Mass increase of 80 Da for G0F / G0F, G1F / G0F, and G1F / G1F (14866 8, 148830, and 148991 Da). To localize the modification, After cleavage of the disulfide bonds with TT, the masses of the light and heavy chains were measured. No difference was observed in the heavy chain mass between the fractions and the pooled fraction, indicating that the modifications were located on the heavy chain. As shown in Figure 3, both fractions ( In the stripped and pooled fractions, the light chain apo form mass (23674 Da) and The mass of the glycosylated light chain (23,836 Da) was detected. The peak with an increase of 80 Da in the light chain was Only at 23754 Da was observed in the AEX strip fraction. The mass error of the determination is within 20 ppm. These data indicate that the 80 Da modification significantly reduces the mass of Ab6. This suggests that it is located on the chain.

[0089] Analysis of mAb antibodies by peptide mapping To further identify the modification site, the AEX strip and pool fractions were reduced and alkylated. The peptide mixture was analyzed by Q-TOF MS. Mass mapping was performed. Comparing the UV traces of these two fractions, two differences were observed. As shown in Figure 4(a) and (b), in the AEX strip fraction Two new peaks were detected at retention times of 37.6 and 65.2 minutes. The observed m / z of the peaks was 1165.4796 (2+) at 37.6 min and and 1476.7372(4+)Da at 65.2 min. Light chain peptide AA25-43 with mass errors of 6.4 ppm and 9.8 ppm, respectively + 80Da and AA25-78 + 80Da. -78 contains one miscleavage site. The modified and unmodified forms of 78 are displayed in Figure 4. Extracted ion chromatograms ( Based on the peak area in the SIC, the level of this modified peptide was AA25-43 and 20.9% and 21.6% for AA25-78 compared to their apo forms. It was estimated that.

[0090] MS / MS fragmentation of modified peptides There are two possible modifications that result in an 80 Da increase in mass: phosphorylation (+79. The two modifications are: 1) sulfation (+79.9663 Da) and 2) sulfation (+79.9568 Da). The mass difference between the two is only 0.0095 Da, which makes it difficult to distinguish them by mass alone. First, the target peptide AA25-78 was fragmented by collision-induced dissociation (CID). The resulting fragments were then analyzed by LTQ-Orbitrap MS. As shown in Figure 5, the complete synthesis of the modifier (80 Da) from the precursor ion was confirmed. A complete loss of the peptide was observed. Only fragments from the peptide backbone were detected, which corresponded to the LC2 The peptide sequence of 5-43 was confirmed. No specific information was obtained. Sulfated tyrosine (sY) is highly unstable and It has been reported that it can be easily lost under standard CID conditions (Nemeth-Cawley y JF1,KS,Rouse JC.,Analysis of sulfate d peptides using positive electrospray i onization tandem mass spectrometry.J Mas Spectrom., 2001.36(12):p.1301-11). Peptide-based Because the original precursor ion was not present at the time of chain fragmentation, the positive ion CI It is not possible to obtain site-specific information regarding the location of the sulfate moiety using D MS / MS. On the other hand, phosphopeptides tended to survive CID, and the peptide backbone Fragmentation of the nucleoside allows for site-specific identification of modifications (Nemeth-Cawl ey JF1,KS,Rouse JC.,Analysis of sulfat ed peptides using positive electrospray ionization tandem mass spectrometry.J Ma ss Spectrom., 2001.36(12):p.1301-11). A neutral loss of 80 Da from the precursor ion was observed. The neutral loss ion is -H3PO4 (-98 Da), and the characteristic fragment ion is P O3 - (-79 Da). On the other hand, sulfation leads to a characteristic neutral loss event. Both the onion and fragment ion are -SO3 ions with 80 Da (Moni gatti F, HB, Steen H., Protein sulfation analysis--A primer.Biochim Biophys Acta. ,2006.1764(12):p.1904-13). CID MS2 data is 80 This suggests that the Da modification is sulfation.

[0091] Another widely used fragmentation mechanism is electron transfer dissociation (E TD), which transfers electrons to multiply protonated peptides / proteins, which is N -Cα backbone bond cleavage, resulting in c-type and z-type cleavage without loss of PTM localization information It can generate type fragment ions (Mikesh LM, UB, Chi A, Co on JJ, Syka JE, Shabanowitz J, Hunt DF.,The utility of ETD mass spectrometry in pro teomic analysis.Biochim Biophys Acta.,20 06.1764(12):p.1811-22). ETD provides complementary information together with CID. That is, the ETD method allows the retention of the SO3 group and therefore the amino acid localization. CID allows for the synthesis of labile modifications, while CID preferably fragments labile modifications. In our case, we used LTQ-O with ETD fragmentation and high-resolution mass detection. The target peptides were analyzed by rbitrap (orbitrap). As shown in Fig. 1, partial loss of the 80 Da modification was observed in the precursor ion. The fragment ions of SO3 bonded to the 80 Da atom are shown. Based on the detection of on (c9, c11, c12-16), the modification site was tyrosine 3 on the light chain. 1, which resides within the CDR1 region of the mAb molecule.

[0092] Alkaline phosphatase treatment Tyrosine phosphorylation and sulfation are isobaric and therefore alkaline Phosphatases were used to distinguish between these two modifications (Yu Y, HA, Moore e KL,Leary JA.,Determination of the site s of tyrosine O-sulfation in peptides an dproteins.Nat Methods,2007.4(7):p.583-8 Alkaline phosphatase is widely used to remove phosphorylation groups from proteins. Chicken albumin was used as a positive control, and this protein The oxidized and glycosylated forms of AEX are well known. The chicken albumin and mAb in the fractions were treated with phosphatase and incubated in parallel at 37°C. Figure 7 shows the results of the mAb and chicken IgG before and after phosphatase treatment. 7(a) shows the measured intact mass of chicken ovalbumin. As shown in FIG. No mass change was observed for Ab. On the other hand, for chicken albumin, 16 A clear mass shift of 0 Da was observed for all major glycoforms (Figure 7(b)). )). Chicken albumin contains two phosphorylation sites, so the loss of 160 Da is This demonstrates the activity of alkaline phosphatase. No phosphorylation was observed, indicating that this mAb is not phosphorylated in the AEX strips. It is suggested that...

[0093] Western blot Thus far, LC / MS analysis has demonstrated that the 80 Da addition to tyrosine 31 on the light chain CDR The mAb AEX strips after phosphatase treatment were used to investigate the properties of the mAb. MS2 and mass spectrometry of the triplicate fractions revealed that the 80 Da adduct was due to sulfation of tyrosine 31. However, these mass spectrometry-based techniques have not been able to identify tyrosine sulfation and The ability to directly distinguish phosphorylation is problematic due to the similar molecular weight of these two groups. To begin to address this issue, we used an anti-sulfotyrosine-specific monoclonal antibody. Western blotting using the same technique was applied to detect the mAb AEX strip fractions. We confirmed the presence of rosin sulfation (Xu J, DX, Tang M, Li L, Xia o L, Yang L, Zhong J, Bode AM, Dong Z, Tao Y, Cao Y.,Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine rec eptor 4 are induced by Epstein-Barr viru s encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.PLoS One.,2013.8(3):p.e56114). In Fig. 8a (upper panel), Normalized concentrations of mAb AEX pool and strip fractions were subjected to reducing SDS PAGE. Western hybridization was used to probe for human heavy and light chains. The heavy and light chains from the pool and strip with added concentrations were separated from the first detection antibody and The tyrosine-specific monoclonal antibody was then used to reprobe the tyrosine-specific ... As shown in Figure 8a (lower panel), the positive signal was It was only detected on the light chain of the knock-in fraction, suggesting that it contains tyrosine sulfation. As a control for cross-reactivity with phosphorylation, lane 1 shows the phosphorylated protein A commercially available extract of EGF-treated A431 cells enriched in α-glutaminase was loaded onto the 100-μg / mL IgG1-containing ... The lack of a positive signal in the IgG4A1 protein indicates that the anti-sulfotyrosine monoclonal antibody does not strongly associate with phosphorylation. The results show that the antibody has no cross-reactivity with the anti-sulfotyrosine antibody (Fig. 8a, lower panel). HEK293 extract suggested by manufacturer as positive control for monoclonal antibodies The product was loaded in lane 2. Positive signals below the bottom 20 KDa were determined according to the manufacturer's This is consistent with the analysis (Figure 8a, lower panel). In Figure 8b, the AEX strip and In addition to the pool, different normalized concentrations of CHO-derived mAbs (mAb1, 2, and 3) were reduced. Human heavy and light chains were analyzed by SDS PAGE and Western hybridization. (top panel), then stripped and probed with anti-sulfotyrosine Consistent with Figure 8a, the tyrosine-probed IgG was reprobed with anti-sulfotyrosine antibody (lower panel). When the AEX mAb strip was placed in the same position, only the AEX mAb strip showed a positive signal at the light chain position. When analyzing the protein, the other three CHO-derived Merck mAbs No positive signal was observed for tyrosine sulfation in these three mice. Increased levels of AEX acidic peaks or tyrosine sulfation hot spots in Ab This is consistent with our observation that no IL-1 was detected (data not shown).

[0094] Comparison of retention times with synthetic peptides with sulfate or phosphorylation To further distinguish between phosphorylation and sulfation, we analyzed either phosphorylation or sulfation on Y31. LC AA25-43 (XSXSXD Y EGDSDXXXXXXX)(distribution A synthetic peptide having the same sequence as that of the synthetic peptide shown in column 65) was analyzed by LC / MS. Peptide XSXSXD Y EGDSDXXXXXXX (SEQ ID NO:65) + phosphorylation, XSX SXD YEGDSDXXXXXXX (SEQ ID NO:65) + Sulfation and AA25-43 + The SIC of 80 Da (in the AEX strip) is shown. The peptides eluted at the same retention time as the AEX strip, but the synthetic peptides with phosphorylation eluted at the same retention time as the AE strip. This is because Y31 on the light chain is sulfated, which is the mechanism behind the present invention. This further proves the observations of the wise.

[0095] Structure of tyrosine sulfation site Protein tyrosine sulfation is carried out by a process called tyrosylprotein sulfotransferase. Previous studies have shown that several acidic residues within the -5 to +5 positions mediate the catalysis of ribonuclease. We showed that TPST recognizes accessible tyrosine residues that are normally surrounded by (Hortin G,FR,Gordon JI,Strauss AW.,Cha racterization of sites of tyrosine sulfa tion in proteins and criteria for predic ting their occurrence.Biochem Biophys Re s Commun.,1986.141(1):p.326-33;Rosenquis t GL,NHJ,Analysis of sequence require ments for protein tyrosine sulfation.Pro tein Sci.,1993.2(2):p.215-22;Teramoto T1 ,FY,Kawaguchi Y,Kurogi K,Soejima M,Ada Chi R, Nakanishi Y, Mishiro-Sato E, Liu MC, Sakakibara Y, Suiko M, Kimura M, Kakuta Y,C rystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism m of protein tyrosine sulfation reaction .Nat Commun.,2013.4:p.1572). The receptor tyrosine is TPS Has a nearby acidic residue to allow recognition of positively charged residues in the T2 substrate binding site The receptor tyrosine is also intrinsically flexible, allowing it to fit into the deep cleft of TPST2. However, the general consensus regarding the site of tyrosine sulfation is unclear. The sequence of sulfated tyrosine has not been determined. The most common features that indicate the vicinity of sulfated tyrosine include: One acidic amino acid within 2 residues of tyrosine, at least 3 acids within 5 residues These include the presence of a turn-inducing amino acid and the presence of a nearby turn-inducing amino acid ( Monigatti F, HB, Steen H., Protein sulfat ion analysis--A primer.Biochim Biophys A cta.,2006.1764(12):p.1904-13). Figure 10 shows the MOE software. Software(Chemical Computing Group,Montreal,C mAb chiro in the context of CDR loops in ribbon diagrams generated by anada The structure of the syn-site is shown. The sequence close to light chain Y31 on this mAb is XSXSXD Y EGDS DXXXXXXX (SEQ ID NO:65). In this sequence, the adjacent residues of Y31, That is, aspartic acid (D) and glutamic acid (E) are both acidic. There are acidic residues, i.e., three D and one E, within five residues of Y31. The turn-inducing residues of Y3, i.e., three serines (S) and one glycine (G), 1. The unique feature of Y31 with adjacent acidic amino acids and local secondary structure elements The structure of plays an essential role in causing this modification.

[0096] The present inventors have demonstrated the unexpected presence of O-linked tyrosine sulfation in CHO-produced antibodies. The position of this labile modification is the site of a protein that undergoes ETD fragmentation. This tyrosine sulfation was found in the CDR1 region of the light chain, as confirmed by quantitative analysis. The results were analyzed by phosphatase treatment, Western blot analysis using anti-tyrosine sulfate antibodies, and This was further confirmed by correlation of retention times with the synthetic sulfated peptides. Structural analysis has demonstrated the influence of acidic residues on sulfation. Secondary structure elements play an essential role in making Y31 a hotspot for sulfation. It is possible that.

[0097] The present invention is to be limited in scope by the specific embodiments described herein. Indeed, the scope of the present invention should not be construed as limited to the embodiments and methods specifically described herein. and other embodiments not specifically described herein, including those specifically described herein. The embodiments listed are not necessarily intended to be exhaustive. Various modifications of the invention in addition to those described above will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the claims.

[0098] Patents, patent applications, publications, product descriptions and protocols are cited throughout this application. No. 6,399,623, the disclosures of which are incorporated herein by reference in their entireties for all purposes. This application is a joint venture of U.S. Provisional Patent Application No. 62 / 414,209, the entirety of which is hereby incorporated by reference. This application claims priority to the present application, which is incorporated herein by reference. All references cited in the article should be linked to individual publications, database entries (e.g. , GenBank sequence or GeneID entry), patent application or patent is Each of the above-mentioned references is incorporated herein by reference to the same extent as if each of the above-mentioned references was specifically and individually indicated to be incorporated herein by reference. This statement of incorporation by reference is intended to 37 C, even if the citation does not immediately precede or follow the sworn statement that it is incorporated by reference. Pursuant to FR § 1.57(b)(1), each individual publication, database entry (e.g. (e.g., Genbank sequence or GeneID entry), patent application or patent [which Each of these shall be clearly identified in accordance with 37 CFR § 1.57(b)(2). It is the applicant's intention to link the present application to the present application, which is incorporated by reference. If a sworn statement is contained in this specification, it is incorporated by reference. Nothing in this specification should be construed as a dilution of this general statement. Citation of a publication is not an admission that the reference is relevant prior art, nor is it intended to be construed as a substitute for the express written consent of the reader. No admission is made as to the contents or dates of these publications or documents. The reference provides definitions for the claimed terms, which are set forth herein. In the event of a conflict with a definition set forth herein, the definition set forth in this application shall govern the interpretation of the claimed invention. This rule shall be used to interpret the

Claims

1. A purified composition comprising a non-CDR-L1 tyrosine sulfated antibody (anti-hLAG3) that specifically binds to human lymphocyte activation gene 3 (hLAG3), the antibody comprising: comprising a heavy chain amino acid sequence of SEQ ID NO:52 and a light chain amino acid sequence of SEQ ID NO:51, and comprising less than 0.5% CDR-L1 tyrosine sulfated anti-hLAG3 antibody compared to a non-CDR-L1 tyrosine sulfated anti-hLAG3 antibody; wherein the CDR-L1 tyrosine sulfated anti-hLAG3 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:52 and a light chain amino acid sequence of SEQ ID NO:51, in which the CDR-L1 tyrosine is sulfated; wherein the non-CDR-L1 tyrosine sulfated anti-hLAG3 antibody and the CDR-L1 tyrosine sulfated anti-hLAG3 antibody are expressed from a mammalian cell; wherein the non-CDR-L1 tyrosine sulfated anti-hLAG3 antibody is a glycosylated antibody species having a molecular weight of 148590 Da, 148749 Da, 148752 Da, 148914 Da, 148915 Da, or a combination thereof; A purified composition.

2. The purified composition of claim 1, comprising a non-CDR-L1 tyrosine-sulfated anti-hLAG3 antibody and a CDR-L1 tyrosine-sulfated anti-hLAG3 antibody in which the N-terminal heavy chain glutamine has been converted to pyroglutamate and / or the C-terminal heavy chain lysine has been removed.

3. The purified composition described in claim 1 or 2, wherein the mammalian cell is a mouse myeloma cell or a hybridoma cell.

4. A purified composition described in any one of claims 1 to 3, wherein the non-CDR-L1 tyrosine sulfated anti-hLAG3 antibody is a glycosylated antibody species having a molecular weight of 148,590 Da, 148,752 Da or 148,914 Da.

5. A non-CDR-L1 tyrosine-sulfated anti-hLAG3 antibody, (i) a molecular weight of 148,590 Da; (ii) G0F glycan, (iii) the N-terminal heavy chain glutamine is converted to pyroglutamate, and (iv) the C-terminal heavy chain lysine has been removed; The purified composition of any one of claims 1 to 3, comprising:

6. A non-CDR-L1 tyrosine-sulfated anti-hLAG3 antibody, (i) a molecular weight of 148749 Da or 148752 Da; (ii) G0F and G1F glycans; (iii) the N-terminal heavy chain glutamine is converted to pyroglutamate, and (iv) the C-terminal heavy chain lysine has been removed; The purified composition of any one of claims 1 to 3, comprising:

7. A non-CDR-L1 tyrosine-sulfated anti-hLAG3 antibody, (i) a molecular weight of 148914 Da or 148915 Da; (ii) G1F glycan, (iii) the N-terminal heavy chain glutamine is converted to pyroglutamate, and (iv) the C-terminal heavy chain lysine has been removed; The purified composition of any one of claims 1 to 3, comprising:

8. The CDR-L1 tyrosine-sulfated anti-hLAG3 antibody is less than 0.5% as compared to a non-CDR-L1 tyrosine-sulfated anti-hLAG3 antibody, as measured by mass spectrometry. The purified composition of any one of claims 1 to 7.

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