US20120196310A1 - A-fucosylation detection in antibodies - Google Patents

A-fucosylation detection in antibodies Download PDF

Info

Publication number
US20120196310A1
US20120196310A1 US13/499,887 US201013499887A US2012196310A1 US 20120196310 A1 US20120196310 A1 US 20120196310A1 US 201013499887 A US201013499887 A US 201013499887A US 2012196310 A1 US2012196310 A1 US 2012196310A1
Authority
US
United States
Prior art keywords
antibody
fragment
endo
residues
analysis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/499,887
Other languages
English (en)
Inventor
Christiane Jaeger
Hans Koll
Peter Sondermann
Pablo Umana
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Glycart AG
Original Assignee
Roche Glycart AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Glycart AG filed Critical Roche Glycart AG
Assigned to ROCHE GLYCART AG reassignment ROCHE GLYCART AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JAEGER, CHRISTIANE, KOLL, HANS, SONDERMANN, PETER, UMANA, PABLO
Publication of US20120196310A1 publication Critical patent/US20120196310A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01096—Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17002—Carboxypeptidase B (3.4.17.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/968—Plasmin, i.e. fibrinolysin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • This invention relates to a method for detecting the presence or absence of fucose residues within a glycosylated antibody or a fragment thereof.
  • variable regions within the Fab (fragment antigen binding) domains of antibodies are responsible for the recognition of the antigen
  • the Fc (fragment crystallizable) region represents an invariant part of the antibody that is responsible for the mediation of effector functions.
  • immunoglobulin G these encompass the fixation of complement and the binding to Fc ⁇ receptors (Fc ⁇ Rs).
  • Fc ⁇ Rs Fc ⁇ receptors
  • the presence of an N-linked oligosaccharide at a single conserved site (Asn297) within the CH2 domain of the homodimeric Fc fragment is mandatory for the mediation of both of these effector functions. It was only recently discovered that modification of the attached carbohydrates can also have an affinity improving effect for the interaction between Fc ⁇ RIIIa and IgG.
  • the carbohydrate modification responsible for this effect is the absence of a fucose residue which is usually attached to the first N-acetylglucosamine (GlcNAc) residue in the biantennary complex-type IgG glycan ( FIG. 1 ).
  • ADCC antibody-dependent cellular cytotoxicity
  • NK natural killer
  • a-fucosylated antibodies represents an important biotechnological challenge which can be achieved by several methods. While cell lines with a complete depletion of enzymes involved in the biosynthesis of fucosylation (e.g. by gene knockout) may yield quantitatively a-fucosylated antibodies, most other methods do not.
  • siRNA treatment or co-cultivation of antibody-expressing cells with kifunensine Zhou et al., Biotechnol Bioeng (2008) 99, 652-665), as well as carbohydrate modification by N-acetylglucosaminyltransferase III (GnT-III), which promotes the formation of bisected oligosaccharides consequently inhibiting the fucosylation reaction (Umana et al., Nat Biotech (1999) 17, 176-180), lead to only partially a-fucosylated antibodies.
  • These partially a-fucosylated antibodies can principally exhibit a heterogeneous a-fucosylation distribution within a pool of antibodies.
  • fucosylation rates can be different during fermentation.
  • the event of fucosylation could be cooperative, i.e. the second fucosylation on the homodimeric antibody may occur with an increased (positive cooperativity) or decreased (negative cooperativity) rate compared to the first one.
  • the Fc ⁇ RIIIa/IgG complex has a 1:1 stoichiometry but IgG has two binding sites for Fc ⁇ RIIIa. Consequently, in a single a-fucosylated antibody the receptor can bind with high affinity to the binding site formed by the IgG's a-fucosylated glycan and protein core or with low affinity to the binding site consisting of the fucosylated carbohydrate and the protein core.
  • a pool of antibodies with 50% a-fucosylation may consist of a homogeneous population of antibodies in which only one of the two N-glycans is fucosylated, or 50% of antibodies in which both N-glycans are fucosylated while in the other 50% none of the N-glycans are fucosylated. It is obvious that such a differential partition of a-fucosylation influences the overall affinity to Fc ⁇ RIIIa and results in a different biological activity. It is therefore mandatory to analyze the biological activity of such an antibody preparation either directly by employing a biological test system (bioassay) or indirectly by biochemically measuring the rate and distribution of the a-fucosylation, which yields a more exact result.
  • bioassay biological test system
  • N-glycosidase F from Flavobacterium meningosepticum to cleave off the N-linked carbohydrates with a subsequent MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analysis (according to Papac et al., Glycobiology (1998) 8, 445-454).
  • MALDI-MS matrix-assisted laser desorption ionization mass spectrometry
  • this invention provides for methods for detecting the presence or absence of fucose residues within a glycosylated antibody.
  • the quantity of fucose residues and their distribution pattern within an antibody or a fragment thereof are determined.
  • the analysis of the distribution of fucose residues per Fc molecule in an antibody preparation is also part of this invention.
  • the present invention can be used for the determination of cooperative fucosylation in an antibody preparation during fermentation.
  • this invention provides for a method that closes a gap in antibody analytics. With the knowledge of fucosylation patterns within an antibody or fragment thereof gained by means of this new method, a more accurate prediction of Fc-mediated potency is now possible.
  • Endo S an enzyme with endoglycosidase activity, originally identified in Streptococcus pyogenes (Collin and Olsen, EMBO J (2001) 20, 3046-3055)
  • Endo S cleaves the complex-type glycan moieties from the Fc region of human IgG, leaving behind just the first GlcNAc residue to which a fucose residue might be attached.
  • the hybrid-type carbohydrates that are discriminated (spared) by Endo S can be quantitatively cleaved at the same site by Endoglycosidase H (Endo H).
  • the present invention relates to a method for detecting the presence or absence of fucose residues within a glycosylated antibody or a fragment thereof.
  • step c) of said method additionally comprises a purification step prior to analysis.
  • purification is achieved by affinity chromatography or size exclusion chromatography. Affinity chromatography can be performed using for example Protein A or Protein G.
  • the protein to be treated and analyzed by the method of the invention is an antibody or an antibody fragment.
  • said antibody is an IgG type antibody.
  • Said antibody fragment is preferably an Fc fragment, in particular an Fc fragment of an IgG type antibody.
  • step a) is performed by one or more enzymes that specifically cleave complex-type or hybrid-type N-linked carbohydrates.
  • these enzymes comprise Endo S and Endo H.
  • step b) is performed by one or more enzymes.
  • these enzymes comprise plasmin and/or carboxypeptidase B.
  • step c) comprises CE-SDS MW (capillary electrophoresis-sodium dodecyl sulfate molecular weight) analysis, ESI-MS analysis or liquid chromatography-mass spectrometry (LC-MS), or a combination thereof.
  • CE-SDS MW capillary electrophoresis-sodium dodecyl sulfate molecular weight
  • ESI-MS analysis electrophoresis-sodium dodecyl sulfate molecular weight
  • LC-MS liquid chromatography-mass spectrometry
  • step a) of the above described method comprises cleavage of the heterogeneous saccharides from the carbohydrate structures of the protein after the first GlcNAc residue of said structures, thereby leaving the fucose residue attached to the antibody core.
  • This step can be performed with two enzymes that specifically cleave complex-type or hybrid-type N-linked carbohydrates that frequently occur in biotechnologically produced antibodies, for example Endo S and Endo H.
  • step b) of the above described method comprises quantitative removal of C-terminal lysine residues of the antibody heavy chain, preferably using an enzyme, said enzyme preferably comprising carboxypeptidase B.
  • step b) of the above described method comprises cleavage between the Fab and the Fc fragment of an antibody.
  • the covalent interchain disulphide bridges within the hinge peptide of the heavy chains are maintained within the Fc-fragment after cleavage between the Fab and the Fc fragment.
  • the cleavage is achieved by an enzyme.
  • such enzyme comprises plasmin.
  • step c) of the above described method comprises analysis of the treated antibody molecule or Fc fragment by LC-MS without any prior purification steps.
  • Such an analysis normally yields only three masses that correspond to proteins with two fucosylated glycans, proteins with one fucosylated and one a-fucosylated glycan, and proteins in which both glycans are a-fucosylated.
  • step c) of the above described method comprises purifying the treated antibody molecule or Fc fragment using standard methods and analyzing it by ESI-MS analysis.
  • ESI-MS analysis normally yields only three masses that correspond to proteins with two fucosylated glycans, proteins with one fucosylated and one a-fucosylated glycan, and proteins in which both glycans are a-fucosylated.
  • the method of the invention comprises the following steps: providing an antibody preparation, optionally isolating the Fc fragment portion of such antibody preparation, removing all heterogeneous saccharide residues from said antibody or Fc fragment with Endo H and Endo S, removing C-terminal lysine residues from said antibody or Fc fragment with carboxypeptidase B, and analysis of the treated antibody or Fc fragment by ESI-MS, LC-MS or CE-SDS MW analysis.
  • said method comprises the following steps: providing an antibody preparation, optionally isolating the Fc fragment portion of such antibody preparation using plasmin, removing all heterogeneous saccharide residues from said antibody or Fc fragment with Endo H and Endo S, removing C-terminal lysine residues from said antibody or Fc fragment with carboxypeptidase B, and purification and analysis of the treated antibody or Fc fragment by ESI-MS, LC-MS or CE-SDS MW analysis.
  • kits suitable for performing an assay which detects the presence or absence of fucose residues within a glycoprotein comprise all components referred to in the methods described above (e.g. Endo H, Endo S, carboxypeptidase B, plasmin, suitable buffers), instructions setting forth a procedure according to any one of the methods described above and a container for the contents of the kit.
  • Endo S for cleavage of complex-type N-linked oligosaccharides of a glycoprotein, preferably a glycosylated antibody or a fragment thereof, is also part of this invention.
  • heterogeneous saccharide includes any monosaccharide moiety of a glycosylated antibody or antibody fragment that is not connected to a fucose residue.
  • Non-limiting examples for heterogeneous saccharides of a glycosylated antibody or antibody fragment are mannose, sialate, galactose, acetylglucosamine.
  • heterogeneous saccharides which are removed in step a) of the method according to the invention will be all saccharides other than the first GlcNAc residue, i.e. the GlcNAc residue attached to an asparagine residue of the protein, and the fucose residue linked to that first GlcNAc residue.
  • heterogenous residues means any other moiety of a glycosylated antibody or antibody fragment (other than heterogenous saccharides) that could interfere with the detection of fucose residues within said antibody or antibody fragment.
  • Non-limiting examples of heterogenous residues are various modifications of the glycosylated antibody or antibody fragment other than fucosylation, such as galactosylation, C-terminal lysine heterogeneity and deamidation.
  • heterogenous residues may further include antibody fragments that are not glycosylated, for example the Fab fragment, the scFv fragment and other fragments.
  • antibody is intended to include whole antibody molecules, antibody fragments, or fusion proteins that include a region equivalent to the Fc region of an immunoglobulin.
  • complex-type oligosaccharide and “hybrid-type oligosaccharide” refer to the glycosylation pattern of an antibody or antibody fragment
  • Non-limiting examples of “complex-type oligosaccharide” and “hybrid-type oligosaccharide” are shown in FIG. 7 .
  • glycoproteins enriched in bisected hybrid-type oligosaccharides typically result from overexpression of GnT-III in production cell lines.
  • Exemplary structures of bisected hybrid-type oligosaccharides are detailed in FIG. 7-III .
  • Glycoproteins enriched in bisected complex type oligosaccharides typically result from a co-expression of ManII and GnT-III in production cell lines.
  • Exemplary structures of bisected, complex-type oligosaccharides are detailed in FIG. 7-IV (Ferrara et al., Biotechnol Bioeng (2006) 93, 851-864
  • Cleavage “after” a sugar residue means cleavage distal to this residue, i.e. cleavage of the sugar bond linking this residue with the adjacent one towards the outer end of the carbohydrate structure.
  • Cleavage “after the first GlcNAc residue” of an N-linked glycan means cleavage of the chitobiose core of the oligosaccharide, between the first (i.e. attached to the asparagine residue) and the second (i.e., attached to the first) GlcNAc residue.
  • “Distribution” of fucose residues within an antibody preparation refers to the presence within that preparation of antibody or Fc molecules differing in the number of fucose residues associated with the N-linked glycans in the Fc region.
  • an IgG molecule has two N-linked glycans in its Fc region, each of which can have a fucose residue linked to the first GlcNAc residue of the carbohydrate structure.
  • IgG with two, one or no fucose residues associated with the N-linked glycans in the Fc region. The ratio of these different species (i.e.
  • the distribution of fucose residues per Fc molecule can be determined by the method of the invention, in addition to determination of the total fucose content, i.e. the fraction of fucosylated or a-fucosylated N-glycans.
  • FIG. 1 Schematic representation of a carbohydrate moiety attached to Asn-297 of human IgG1-Fc.
  • the sugars in bold define the pentasaccharide core of N-linked glycan structures; the addition of the other sugar residues is variable.
  • In grey is represented a bisecting GlcNAc residue.
  • FIG. 2 Deglycosylation of intact Fc fragment of antibodies A (wildtype) and C (glycoengineered) monitored by CE-SDS. Electropherograms of non-reduced Fc fragments are shown before and after enzymatic treatment.
  • A Fc fragment of antibody C without enzymatic treatment (dashed line) and deglycosylated with PNGase F (dotted line) or Endo S (solid line)
  • B Fc fragment of antibody A without enzymatic treatment (dashed line), deglycosylated with PNGase F (dotted line) or deglycosylated with Endo S (solid line).
  • FIG. 3 Positive-ion MALDI-TOF mass spectra of the N-linked oligosaccharides released from Fc fragment of antibody C by consecutive treatment with Endo S and PNGase F or with Endo S and Endo H.
  • A Spectrum of glycans released by treatment with Endo S.
  • FIG. 4 Deglycosylation of the Fc fragment of antibody C monitored by CE-SDS MW analysis (A) and positive-ion MALDI-TOF mass spectrometry (B).
  • A Overlay of electropherogram of the non-reduced Fc fragment without glycosidase treatment (dashed line) and treated with a combination of Endo S and Endo H (solid line).
  • FIG. 5 ESI-MS spectra of Fc fragments after treatment with Endo S and Endo H.
  • A Fc fragments of antibody A
  • B Fc fragments of antibody B
  • C Fc fragments of antibody C. Peak 1: Fc-GlcNAc/GlcNAc
  • Peak 2 Fc-GlcNAc/GlcNAc+Fuc
  • Peak 3 Fc-GlcNAc+Fuc/GlcNAc+Fuc.
  • FIG. 6 Deglycosylation of antibody C monitored by CE-SDS (A) and positive-ion MALDI-TOF mass spectrometry (B).
  • A Electropherograms of non-reduced IgG are shown before and after enzymatic treatment: Antibody C without enzymatic treatment (dashed line) and deglycosylated with PNGase F (dotted line) or combined treatment with Endo S and Endo H (solid line).
  • B Mass spectra of the N-linked oligosaccharides released from entire IgG treated with Endo S and Endo H.
  • FIG. 7 N-linked oligosaccharide biosynthetic pathway leading to complex- or hybrid-type structures.
  • M1 mannosidase I
  • G1 ⁇ 1,2-N-acetylglucosaminyltransferase I
  • G3 ⁇ 1,4-N-acetylglucosaminyltransferase III
  • Gt ⁇ 1,4-galactosyltransferase.
  • FIG. 8 ESI-MS spectra of entire IgGs after treatment with Endo S and Endo H.
  • A antibody A
  • B antibody D. Peak 1: Fc-GlcNAc/GlcNAc
  • Peak 2 Fc-GlcNAc/GlcNAc+Fuc
  • Peak 3 Fc-GlcNAc+Fuc/GlcNAc+Fuc.
  • FIG. 9 LC-MS spectra of entire IgGs after treatment with Endo S and Endo H.
  • A antibody A
  • B antibody D. Peak 1: Fc-GlcNAc/GlcNAc
  • Peak 2 Fc-GlcNAc/GlcNAc+Fuc
  • Peak 3 Fc-GlcNAc+Fuc/GlcNAc+Fuc.
  • the proteins were incubated for 72 hours at 25° C. in 50 mM Tris pH 8.0, 150 mM NaCl with 0.42 U plasmin (Roche) per milligram.
  • Cleaved Fc was separated from Fab-fragments using a Protein A affinity column (5 ml HiTrapTM Protein A HP column, GE Healthcare) equilibrated and washed (5 column volumes (CV)) with buffer A (50 mM Tris pH 8.0, 100 mM glycine, 150 mM NaCl).
  • Fc was eluted by a pH-step using buffer B (50 mM Tris pH 3.0, 100 mM glycine, 150 mM NaCl).
  • Fractions containing Fc were pooled and neutralized by adding 1:40 (v/v) 2 M Tris pH 8.0. Samples were concentrated to a volume of 2.5 ml using ultra concentrators (Vivaspin 15R 10'000 MWCO HY, Sartorius) and subsequently applied to a PD-10 desalting column (GE Healthcare) equilibrated with 2 mM MOPS pH 7.4, 150 mM NaCl, 0.02% (w/v) NaN 3 . Purified protein was frozen in liquid nitrogen and stored at ⁇ 80° C.
  • N-linked glycans of human IgG Different enzymes were used for hydrolyzing the N-linked glycans of human IgG.
  • the N-linked oligosaccharides were cleaved from 1 mg of Fc by incubation with 0.005 U recombinant PNGase F (QAbio, Vista Monte, USA).
  • PNGase F a recombinant PNGase F
  • samples were incubated with either a molar ratio of 1:20 of Endo S alone or in combination with 0.1 U/mg Endo H (QAbio). All reactions were incubated in 20 mM Tris pH 8.0 at 37° C. for 16 h.
  • Fc was purified after Endo S treatment by affinity chromatography using Protein A and subsequently digested with either PNGase F or Endo H, as described above.
  • the N-linked glycans of human IgG were released using different enzymes.
  • the N-linked oligosaccharides were cleaved from 1 mg of IgG by incubation with 0.005 U of recombinant PNGase F (QAbio) in 20 mM Tris pH 8.0 at 37° C. for 16 h.
  • PNGase F recombinant PNGase F
  • samples were applied to a NAP-5 desalting column (GE Healthcare) equilibrated with 20 mM Tris pH 8.0.
  • Eluted sample was concentrated to a final concentration of 4 mg/ml using ultra concentrators (Amicon 5′000 MWCO, Millipore) and incubated with a molar ratio of 1:7 of Endo S combined with 0.1 U/mg Endo H (QAbio) at 37° C. for 16 h.
  • carboxypeptidase B (Roche; 1 mg/ml). Therefore 1 ⁇ l carboxypeptidase B per 50 ⁇ g human Fc or entire antibody was added to the Endoglycosidase reaction and incubated again for 1 h at 37° C.
  • Neutral oligosaccharide profiles of the human Fc or entire antibody were analyzed by mass spectrometry (Autoflex, Bruker Daltonics GmbH) in positive ion mode (Papac et al., 1998).
  • Fc or entire IgG was separated from enzymes and cleaved carbohydrates by Protein A affinity chromatography using Agilent HPLC 1100 series (Agilent Technologies). Samples were applied to Protein A matrix (Poros 20 A; Applied Biosystems) packed in a guard column 2 ⁇ 20 mm C-130B (Upchurch Scientific) equilibrated with buffer A (50 mM Tris, 100 mM glycine, 150 M NaCl, pH 8.0). After washing with 5.5 CV of buffer A, human Fc or entire IgG was eluted by a pH-step using buffer B (50 mM Tris, 100 mM glycine, 150 M NaCl, pH 3.0) over 8.3 CV. The fraction containing the protein was neutralized by adding 1:40 (v/v) 2 M Tris pH 8.0.
  • buffer A 50 mM Tris, 100 mM glycine, 150 M NaCl, pH 3.0
  • the purified protein was subsequently further used for either treatment with enzymes to analyze non-cleaved carbohydrates, CE-SDS analysis or ESI-MS.
  • the buffer of Protein A purified samples was exchanged to 2 mM MOPS pH 7.4, 150 mM NaCl, 0.02% (w/v) NaN 3 using spin concentrators (5000 MWCO, Millipore). Proteins were frozen in liquid nitrogen and stored at ⁇ 80° C.
  • the injected protein sample was desalted applying an 8 minute isocratic elution with 2% formic acid, 40% acetonitrile at a flow rate of 1 ml/min.
  • the elution of the desalted protein was recorded by UV at 280 nm and the eluting sample (volume about 200-300 ⁇ l) was collected in a 1.5 ml reaction vial.
  • MS spectra were acquired using a capillary voltage of 1000 V, a cone voltage of 30 V in a mass range from 1000-2000 m/z in positive ion mode using a source temperature of 80° C. Desolvation temperature was off. MS data were acquired for approx 2-3 minutes by the respective instrument software.
  • MS spectra were acquired using a NanoMate device as spray interface.
  • the values for data acquisition at the MS instrument were set to 400 Vpp (funnel RF), 120 eV (ISCID energy) and 400 Vpp (Multipol RF) regarding the transfer parameters, 5.0 eV (ion energy) and 300 m/z (low mass) for the quadrupol parameters, 15 eV (collision energy) and 3000 Vpp (collision RF) adjusting the collision cell and 800 Vpp, 160 ⁇ s for transfer time and 20 ⁇ s prepulse storage at the ion cooler. Data were recorded at a mass range from 1000-4000 m/z in positive ion mode.
  • Molar masses of dimeric Fc-fragments and entire antibody, containing different combinations of glycan structures truncated by the endoglycosidases applied i.e. GlcNAc/GlcNAc, GlcNAc+Fuc/GlcNAc and GlcNAc+Fuc/GlcNAc+Fuc, were determined from the respective m/z pattern of the Fc fragment or entire antibody species using an in-house developed software.
  • the relative ratios of the various residually glycosylated dimeric Fc fragments or entire antibodies were calculated with the same in-house software using the sum of peak areas of the m/z spectrum of a distinct glycosylation variant of the dimeric Fc-fragment or entire antibody.
  • LC-MS was performed on a Dionex HPLC system (Dionex Ultimate 3000) coupled to a Q-TOF II mass spectrometer (Waters).
  • the chromatographic separation was performed on a ACE C4 column (5 ⁇ m particle size, 300 A pore size, 1 ⁇ 30 mm; Advanced Chromatography Technologies).
  • Eluent A was 0.1% formic acid
  • eluent B was 99.9% acetonitrile and 0.1% formic acid.
  • the flow rate was 100 ⁇ l/min, the separation was performed at 75° C. and 2 ⁇ g (10 ⁇ l) of an intact antibody sample treated with Endo S and Endo H, but without plasmin treatment, were used.
  • MS spectra were acquired using a capillary voltage of 2700 V, a cone voltage of 80 V in a mass range from 1000-4000 m/z in positive ion mode using a source temperature of 100° C. Desolvation temperature was set to 200° C. MS data were acquired for approximately 11.4 minutes (gradient time 3.5 to 14.9 min) by the respective instrument software.
  • Molar masses of intact antibody consisting of two heavy chains and two light chains
  • glycan structures truncated by the endoglycosidases applied i.e. GlcNAc/GlcNAc, GlcNAc+Fuc/GlcNAc and GlcNAc+Fuc/GlcNAc+Fuc, were determined from the respective m/z pattern of the antibody species using an in-house developed software.
  • the relative ratios of the various residually glycosylated intact antibodies were calculated with the same in-house software using the sum of peak areas of the m/z spectrum of a distinct glycosylation variant of the intact antibody.
  • the ratio of non-fucosylated heavy chains was determined by reducing the EndoS and EndoH-treated antibody with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) and performing an LC-MS analysis as described before, using the same column type and gradient setting but some modified parameters for MS data acquisition. MS parameters were the same as described before, but cone voltage was set to 25 V and mass range was from 600-2000 m/z.
  • N-Glycosidase F also known as PNGase F
  • PNGase F is a highly specific deglycosidase that cleaves between the innermost N-acetylglucosamine of high mannose-, hybrid-, and complex-type N-linked oligosaccharides and the asparagine residue of the glycoprotein to which the glycan is attached (Tarentino et al., 1985).
  • Treatment of the Fc fragments of antibody A and C with PNGase F according to the instructions of the manufacturer was monitored by CE-SDS. Under these conditions PNGase F quantitatively removes the glycan moiety of both analyzed samples, resulting in a mobility shift of the main peak from 3.79 ⁇ 10 ⁇ 5 to 3.9 ⁇ 10 ⁇ 5 ( FIG. 2 ).
  • Endo S cleaves the chitobiose core of N-linked oligosaccharides, leaving the first N-acetylglucosamine residue—and an ⁇ -fucose residue in case of fucosylated carbohydrates—attached to the protein.
  • the CE-analysis of a such digested glycoengineered sample revealed that approximately 10% of the protein were still undigested ( FIG. 2 a , Table 2), as demonstrated by a peak with a mobility of 3.84 ⁇ 10 ⁇ 5 .
  • Subsequent analysis by PNGase F treatment indicated that the Endo S resistant carbohydrates were entirely of hybrid structure suggesting specificity of this enzyme for complex carbohydrates. This result could be corroborated by the quantitative Endo S digestion of wildtype antibody A which resulted in homogenously deglycosylated protein ( FIG. 2 b ).
  • Peak area of enzyme-treated Fc fragments evaluated by CE-SDS Peak area [%] Antibody, enzyme Non-cleaved Cleaved A, no enzyme 99.3 0.7 A, PNGase F 1.3 98.7 A, Endo S 1.8 98.2 C, no enzyme 100.0 0.0 C, PNGase F 0.3 99.7 C, Endo S 10.6 89.4
  • Endo S-treated Fc of antibody C was purified by affinity chromatography to remove the enzyme and cleaved carbohydrates, and subsequently incubated with PNGase F to remove the entire glycan moiety.
  • Endo S is a recombinant glycosidase that cleaves within the chitobiose core of high mannose- and hybrid-type N-linked oligosaccharides of glycoproteins. It is not able to cleave within complex structures.
  • ESI-MS spectra revealed Fc fragments with either two, one or no fucose linked to the residual GlcNAc still attached to the protein after EndoS/EndoH treatment ( FIG. 5 ).
  • Distribution of these three fucose species is summarized for the investigated three different IgGs A, B and C (calculated as relative ratio of the sum of peak areas in the m/z-spectra). The results correlate well with the fucose content determined by MALDI-TOF MS (Table 3).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US13/499,887 2009-10-02 2010-09-28 A-fucosylation detection in antibodies Abandoned US20120196310A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP09172130.8 2009-10-02
EP09172130 2009-10-02
PCT/EP2010/064291 WO2011039150A1 (en) 2009-10-02 2010-09-28 A-fucosylation detection in antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2010/064291 A-371-Of-International WO2011039150A1 (en) 2009-10-02 2010-09-28 A-fucosylation detection in antibodies

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/285,440 Continuation US20140302544A1 (en) 2009-10-02 2014-05-22 A-fucosylation detection in antibodies

Publications (1)

Publication Number Publication Date
US20120196310A1 true US20120196310A1 (en) 2012-08-02

Family

ID=43037129

Family Applications (3)

Application Number Title Priority Date Filing Date
US13/499,887 Abandoned US20120196310A1 (en) 2009-10-02 2010-09-28 A-fucosylation detection in antibodies
US14/285,440 Abandoned US20140302544A1 (en) 2009-10-02 2014-05-22 A-fucosylation detection in antibodies
US15/179,431 Active 2030-12-24 US10352943B2 (en) 2009-10-02 2016-06-10 A-fucosylation detection in antibodies

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14/285,440 Abandoned US20140302544A1 (en) 2009-10-02 2014-05-22 A-fucosylation detection in antibodies
US15/179,431 Active 2030-12-24 US10352943B2 (en) 2009-10-02 2016-06-10 A-fucosylation detection in antibodies

Country Status (8)

Country Link
US (3) US20120196310A1 (zh)
EP (1) EP2483693B1 (zh)
JP (2) JP2013506404A (zh)
CN (1) CN102511007B (zh)
CA (1) CA2773882C (zh)
ES (1) ES2528321T3 (zh)
HK (1) HK1168653A1 (zh)
WO (1) WO2011039150A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140081813A (ko) * 2011-09-13 2014-07-01 게노비스 에이비 스트렙토코쿠스 피오게네스 유래의 엔도글리코시다아제 및 그의 이용방법
US10858641B2 (en) 2016-02-08 2020-12-08 Synaffix B.V. Enzymes for trimming of glycoproteins

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MY169935A (en) * 2011-04-29 2019-06-18 Biocon Biologics India Ltd "a method for reducing heterogeneity of antibodies and a process of producing the antibodies thereof"
WO2013057078A1 (en) 2011-10-19 2013-04-25 Roche Glycart Ag Separation method for fucosylated antibodies
EP2812442B9 (en) 2012-02-10 2023-02-15 University of Maryland, Baltimore Chemoenzymatic glycoengineering of antibodies and fc fragments thereof
TWI682033B (zh) * 2014-03-17 2020-01-11 泉盛生物科技股份有限公司 製造具有經修飾的糖苷化作用之重組糖蛋白之方法
JP6750148B2 (ja) * 2014-04-25 2020-09-02 公益財団法人野口研究所 糖鎖切断抗体の製造方法及び均一糖鎖抗体
EP3064507A1 (en) 2015-03-06 2016-09-07 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Fusion proteins comprising a binding protein and an interleukin-15 polypeptide having a reduced affinity for IL15ra and therapeutic uses thereof
CN108982737B (zh) * 2018-08-07 2021-06-22 中国人民解放军军事科学院军事医学研究院 一种规模化的核心岩藻糖基化修饰位点占有率定量方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5126250A (en) * 1988-09-28 1992-06-30 Eli Lilly And Company Method for the reduction of heterogeneity of monoclonal antibodies

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4253233B2 (ja) * 2003-08-25 2009-04-08 大塚製薬株式会社 術後癌患者の予後判定方法
US8207303B2 (en) * 2005-02-18 2012-06-26 Medarex, Inc. Monoclonal antibodies against CD30 lacking in fucosyl residues
CN101238148A (zh) * 2005-06-03 2008-08-06 健泰科生物技术公司 具有改良的岩藻糖化水平的抗体的生产方法
US20070166306A1 (en) * 2006-01-17 2007-07-19 Fey Georg H M Anti-CD19 antibody composition and method
CN101460522B (zh) * 2006-04-11 2014-07-23 弗·哈夫曼-拉罗切有限公司 糖基化抗体
US20080226635A1 (en) * 2006-12-22 2008-09-18 Hans Koll Antibodies against insulin-like growth factor I receptor and uses thereof
JP5175336B2 (ja) 2007-04-16 2013-04-03 モメンタ ファーマシューティカルズ インコーポレイテッド 細胞表面グリコシル化に関連する方法
WO2009033670A2 (en) 2007-09-14 2009-03-19 Genovis Ab Method and kits for dissociating fcgamma-receptor-igg complexes and for igg purification and detection

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5126250A (en) * 1988-09-28 1992-06-30 Eli Lilly And Company Method for the reduction of heterogeneity of monoclonal antibodies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Collin, M et al. EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. 2001. The EMBO Journal. 20:3046-3055. *
Huhn C et al. IgG glycosylation analysis. 2009. Proteomics. 9, 882-913. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140081813A (ko) * 2011-09-13 2014-07-01 게노비스 에이비 스트렙토코쿠스 피오게네스 유래의 엔도글리코시다아제 및 그의 이용방법
US20140302519A1 (en) * 2011-09-13 2014-10-09 Genovis Ab Endoglycosidase from streptococcus pyogenes and methods using it
US9493752B2 (en) * 2011-09-13 2016-11-15 Genovis Ab Endoglycosidase from Streptococcus pyogenes and methods using it
KR102001427B1 (ko) 2011-09-13 2019-07-18 게노비스 에이비 스트렙토코쿠스 피오게네스 유래의 엔도글리코시다아제 및 그의 이용방법
US10858641B2 (en) 2016-02-08 2020-12-08 Synaffix B.V. Enzymes for trimming of glycoproteins
US11713453B2 (en) 2016-02-08 2023-08-01 Synaffix B.V. Enzymes for trimming of glycoproteins

Also Published As

Publication number Publication date
CN102511007A (zh) 2012-06-20
EP2483693A1 (en) 2012-08-08
US10352943B2 (en) 2019-07-16
ES2528321T3 (es) 2015-02-06
JP2015109857A (ja) 2015-06-18
JP5980353B2 (ja) 2016-08-31
US20170108510A1 (en) 2017-04-20
CA2773882A1 (en) 2011-04-07
CA2773882C (en) 2016-01-12
WO2011039150A1 (en) 2011-04-07
JP2013506404A (ja) 2013-02-28
HK1168653A1 (zh) 2013-01-04
EP2483693B1 (en) 2014-12-03
US20140302544A1 (en) 2014-10-09
CN102511007B (zh) 2015-04-15

Similar Documents

Publication Publication Date Title
US10352943B2 (en) A-fucosylation detection in antibodies
Flynn et al. Naturally occurring glycan forms of human immunoglobulins G1 and G2
Hafkenscheid et al. Structural analysis of variable domain glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis reveals the presence of highly sialylated glycans
Chen et al. Analysis of N-glycans from recombinant immunoglobulin G by on-line reversed-phase high-performance liquid chromatography/mass spectrometry
US20180164325A1 (en) Immunoglobulin glycosylation pattern analysis
Wang et al. Antibody glycosylation: impact on antibody drug characteristics and quality control
Sinha et al. Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs
Prater et al. High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry
US20180265544A1 (en) Separation method for fucosylated antibodies
Maier et al. Applying mini-bore HPAEC-MS/MS for the characterization and quantification of Fc N-glycans from heterogeneously glycosylated IgGs
Goetze et al. Rapid LC–MS screening for IgG Fc modifications and allelic variants in blood
Shrivastava et al. N-Glycosylation of monoclonal antibody therapeutics: A comprehensive review on significance and characterization
Ip et al. Structural characterization of the N-glycans of a humanized anti-CD18 murine immunoglobulin G
Triguero et al. Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants
Messina et al. HILIC-UPLC-MS for high throughput and isomeric N-glycan separation and characterization in Congenital Disorders Glycosylation and human diseases
US20140070086A1 (en) Method for analyzing glycan structure
Montesino et al. Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development
Freimoser–Grundschober et al. FcγRIIIa chromatography to enrich a-fucosylated glycoforms and assess the potency of glycoengineered therapeutic antibodies
Kondo et al. From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples
Séveno et al. Plant N-glycan profiling of minute amounts of material
US20210062156A1 (en) Antibodies with modulated glycan profiles
US20220177582A1 (en) Non-consensus glycosylation of bispecific antibodies
Kurahashi et al. Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS
Cao et al. Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards
US20150210777A1 (en) Glycoprotein

Legal Events

Date Code Title Description
AS Assignment

Owner name: ROCHE GLYCART AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JAEGER, CHRISTIANE;KOLL, HANS;SONDERMANN, PETER;AND OTHERS;REEL/FRAME:028412/0035

Effective date: 20100817

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION