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US20050287628A1 - System and method for the production of recombinant glycosylated proteins in a prokaryotic host - Google Patents

System and method for the production of recombinant glycosylated proteins in a prokaryotic host Download PDF

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US20050287628A1
US20050287628A1 US10/506,917 US50691705A US2005287628A1 US 20050287628 A1 US20050287628 A1 US 20050287628A1 US 50691705 A US50691705 A US 50691705A US 2005287628 A1 US2005287628 A1 US 2005287628A1
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proteins
prokaryotic organism
oligosaccharide
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Markus Aebi
Michael Wacker
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Priority to US12/219,383 priority patent/US8703471B2/en
Priority to US14/189,673 priority patent/US20140335127A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an expression system and a method for the production of recombinant human and/or animal and/or plant and/or prokaryotic and/or fungal glycoproteins.
  • Such glycoproteins may serve as nutrition or medical drugs for human or animals or plants because of their identical structure to the glycoproteins normally produced in these organisms.
  • Glycosylation constitutes one of the most important of all post-translational protein modifications in eukaryotic cells and may have numerous effects on function, structure, physical properties and targeting of particular proteins.
  • carbohydrate moiety is to be regarded as having significant effects on both the structure and on the physicochemical features of a protein and may affect its enzymatic activity, antigenicity or thermal stability.
  • the sugars can be linked via the ⁇ -amine group of an asparagine (N-glycosidic bond) or the hydroxyl group of a serine or threonine (O-glycosidic bond) residue.
  • the N-linked protein glycosylation Is by far the most common protein modification found in eukaryotes.
  • the complex glycosylation process starts at the cytoplasmic face of the endoplasmatic reticulum (ER) with the assembly of an oligosaccharide on the lipid carrier dolichylpyrophrsphate [Burda, P. and Aebi, M, (1999) The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta, 1426, 239-257]: 2 N-acetylglucosamine and 5 mannose residues are attached to this lipid in a stepwise fashion.
  • the lipid linked oligosaccharide (LLO) is then flipped into the lumen of the ER, where by addition of 4 mannose and 3 glucose residues full length LLO is obtained in the central reaction of the process, this oligosaccharide is transferred to selected asparagine residues of newly synthesized polypeptides.
  • This reaction is catalyzed by the oligosaccharyl transferase (OTase) in the lumen of the ER.
  • OTase is a complex of at least 8 subunits and this enzyme is responsible for the formation of the N-glycosidic bond. While still in the ER, three glucose and one mannose residue are quickly removed from the oligosaccharide of the glycoprotein.
  • Glycoproteins are then transported to the Golgi apparatus where further trimming and addition of sugar moieties occurs before they are targeted to their final destinations [Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G. and Marth, 3 . (1999) Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.].
  • LLO synthesis is a highly conserved process in eukaryotic cells
  • the modifications in the Golgi are not only species specific but also cell-type specific and lead to a high degree of diversity with respect to the structure of the N-linked oligosaccharides.
  • heterologous expression systems The production of human proteins in a variety of heterologous expression systems has become an important technique to generate recombinant proteins for research purposes as well as pharmaceutical applications. It is generally recognized that there is no universal expression system available for production. Furthermore, the selection of a cell type for expression of heterologous proteins depends on many factors. These include criteria such as cell growth characteristics, expression levels, intracellular or extracellular expression, and biological activity of the protein of interest as well as its intended use. But one of the most important criteria to be considered is whether a protein needs to be glycosylated for its application. Many human therapeutics are glycoproteins, and the importance of the posttranslational modification of polypeptides with defined oligosaccharides is well documented by their implication in numerous biological phenomena.
  • N-glycans In mammalian glycoproteins, the majority of N-glycans are of the complex type, i.e. they consist of a pentasaccharide core of two N-acetylglucosamine and three mannose residues. This core is the remaining structure of the oligosaccharide that was originally transferred from dolichylpyrophosphate to proteins. In the Golgi it is further modified with antennae comprising additional N-acetylglucosamine, galactose, sialic acid and often fucose residues. An enormous diversity of impressively complex oligosaccharide structures is thereby possible.
  • CDG congenital disorder of glycosylation
  • glycosylated proteins Since the majority of therapeutically relevant proteins are glycosylated in their natural forms, they should also be glycosylated as recombinant proteins in order to get the correct biological activity. Thus, monitoring of the glycosylation pattern in quality control of recombinant proteins to assure product safety, efficiency and consistency has become increasingly important.
  • Systems for the expression of glycosylated proteins have been developed. The most commonly used are Chinese hamster ovary (CHO) cell lines [Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M. and Conradt, H. S. (1999) Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells.
  • Glycoconjugate Journal 16, 81-97] insect cells [Altmann, F., Staudacher, E., Wilson, I. B. and Marz, L. (1999) Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconjugate Journal, 16, 109-1231 or fungal cells [Malissard, M., Zeng, S. and Berger, E. G. (1999) The yeast expression system for recombinant glycosyltransferases. Glycoconjugate Journal, 16, 125-139. or Maras, M., van Die, i., Contreras, R. and van den Hondel, C. A.
  • Insect cells are also widely used to produce recombinant proteins, as they can synthesize large quantities of a protein of interest when infected with powerful baculovirus-based gene expression vectors, and they can provide post-translational modifications similar to those provided by mammalian cells.
  • the N-glycosylation pathway parallels the mammalian pathway until the formation of the core pentasaccharide.
  • normally insect cells do not express additional transferases in the Golgi and therefore the tv-glycans produced are truncated (paucimannosidic) instead of a complex type as found in mammalian cells.
  • Fungi in particular Saccharomyces cerevisiae or Pichia pastoris, are suitable host organisms for the production of eukaryotic heterologous proteins.
  • These systems combine well-known techniques for the molecular and genetic manipulations, the cells are easy to grow and they have the capability for complex post-translational modifications such as protein glycosylation.
  • fungi do not further trim the oligosaccharide in the Golgi but instead elongate it directly by the addition of mannose residues to form mannanes with up to 200 mannose units. Some glycoproteins escape these modifications and their maturation is more limited, yielding short core type oligosaccharides with up to 13 mannose residues.
  • N-glycosylation in eukaryotes emphasize the differences in the structure of N-glycans. The implication on the function reveals that exact analysis of the structure is essential. Significant advances in carbohydrate structural analyses have been achieved during the past years. Especially in mass spectrometry (on-line ESI-MS, nanospray tandem mass spectrometry (ESI-MS/MS) and improved MALDI/TOF techniques), very sensitive instrumentation for glycosylation analysis has been made available.
  • mass spectrometry on-line ESI-MS, nanospray tandem mass spectrometry (ESI-MS/MS) and improved MALDI/TOF techniques
  • Baculoviruses essentially have a lytic infection mode, i.e. when the product is harvested, a large proportion of the host cells is lysed and releases degradative enzymes.
  • the protein synthesis is maximal near death of infected cells and it is possible that the overall processing of the protein is suboptimal at that time.
  • Particularly proteins destined for the plasma membrane or for secretion are affected by the depletion of components of the post-translational machinery of the secretary pathway.
  • large scale insect cell culture offers particular challenges to the biotechnologist due to the higher oxygen consumption and higher shear sensitivity of the cells as compared to mammalian cells.
  • glycoproteins Like in mammalian cells, the major drawback in the heterologous expression of glycoproteins resides in the different structure of the N-alycan as described before, Especially the lack of terminal sialic acid residues is detrimental, because these sugars play important roles in glycoprotein biology.
  • the three main eukaryotic expression systems mostly fall to produce glycans of a desired structure.
  • the gram-negative bacterium Escherichia coli offers several technical advantages for the production of heterologous proteins. It is the oldest and most productive system used, However, the inability of E. coli cells to exert post-translational modifications of proteins remains the strongest drawback for its use as the preferred host for the production of human proteins.
  • This object of the invention is reached—according to a first aspect—by the combination of features of independent claim 1 , wherein a system is proposed for the production of recombinant human, human-like, or animal, or plant, or fungal, or bacterial N-glycosylated target proteins, the system comprising a prokaryotic organism into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N-glycosylation of the target protein.
  • the system according to the invention is characterized in that said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins.
  • This object of the invention is reached—according to a second aspect—by the combination of features of independent claim 5 , wherein a method is proposed for producing recombinant human, human-like, or animal, or plant, or fungal, or bacterial N-glycosylated target proteins, the system comprising a prokaryotic organism into which is introduced a genetic information encoding for a metabolic apparatus capable of carrying out the requested N-glycosylation of the target protein.
  • the method according to the invention is characterized in that said prokaryotic organism also contains the genetic information required for the expression of one or more recombinant target proteins.
  • E. coli Since E. coli is easier to handle and to grow and its genetics are very well known, the production of human, human-like, animal or plant or fungal or bacterial glycoproteins in E. coli is a breakthrough in biotechnology.
  • N-glycans of recombinant glycoproteins depend on the glycosylation genes present in the expression system used.
  • FIG. 1 the expression of recombinant glycoproteins in eukaryotes
  • FIG. 1A shows the expression of a target glycoprotein
  • FIG. 1B shows genetic engineering of existing glycosylation pathways in the Golgi
  • FIG. 2 the Escherichia coli expression system, with the expression of a recombinant target protein and the introduction of a specific glycosylation pathway according to the invention
  • FIG. 3 the legend for the signs representing individual elements of the oligosaccharides residues of the glycoproteins in FIG. 1 and FIG. 2 .
  • FIG. 1 shows the expression of recombinant glycoproteins in eukaryotic expression systems.
  • FIG. 1A shows the expression of a target glycoprotein, wherein the assembly of the lipid linked oligosaccharide (LLO; step I) and the transfer of the oligosaccharide to the protein by means of an OTase (step II) is a highly conserved process in the Endoplasmatc Reticulum (ER).
  • LLO lipid linked oligosaccharide
  • step II the transfer of the oligosaccharide to the protein by means of an OTase
  • the modifications in the Golgi are cell type specific (step III).
  • FIG. 1B again shows the expression of a target glycoprotein, wherein the assembly of the lipid linked oligosaccharide (LLO; step I) and the transfer of the oligosaccharide to the protein by means of a OTase (step II) is a highly conserved process in the ER.
  • LLO lipid linked oligosaccharide
  • step II OTase
  • FIGS. 1A and 1B show that the expression of the recombinant protein is carried out outside the ER (step Ib) and that this target protein then is imported into the ER (step IIb).
  • the explanation of the signs representing the individual elements of the oligosaccharides derives from the legend in FIG. 3 .
  • This solution offers the possibility to design the oligosaccharide structure by the expression of specific glycosyltranferases and does not affect vital functions of the production cell.
  • FIG. 2 shows the Escherichia coli expression system according to the invention with the expression of a recombinant target protein (step Ib), which then is introduced to the glycoprotein synthesis (step IIb).
  • step Ib a recombinant target protein
  • step IIb glycoprotein synthesis
  • specific glycosyltransferases for the assembly of the lipid-linked oligosaccharide (LLO′′; step !) are introduced into the host.
  • the OTase covalently links this oligosaccharide to specific residues of the desired protein (step II).
  • the oligosaccharide that is attached to the desired protein as described in FIG. 2
  • Endo-A immobilized endo- ⁇ -N-acetylglucosaminidase from Arthrobacter protophormiae
  • ribonuclease B that contained a covalently linked N-acetylglucosamine
  • the invention encompasses the production of glycosylated glycoproteins.
  • benefits include, but are not limited to, increased in vivo circulatory half life of a protein; increased yields of recombinant proteins; increased biological activity of the protein including, but not limited to, enzyme activity, receptor activity, binding capacity; altered antigenicity; improved therapeutic properties; increased capacities as a vaccine or a diagnostic tool, and the like.
  • mammalian glycoproteins that can be produced with this invention and that can serve as medicaments for humans, animals or plants, include but are not limited to, erythropoietin, transferrin, interferons, immunoglobulines, interleukins, plasminogen, and thyrotropin.
  • prokaryotic and/or fungal glycoproteins can be produced with the invention and can serve as medicaments for humans, animals and plants, e.g. glycoproteins from C. jejuni and from fungi.
  • Further applications for glycoproteins produced with this invention include, but are not limited to, industrial enzymes, functional food, cosmetics, packaging materials, and textiles.
  • the present invention bases on the finding, that Campylobacter jejuni, a gram-negative bacterium, produces glycoproteins.
  • Campylobacter jejuni a gram-negative bacterium
  • the present invention bases on the finding, that Campylobacter jejuni, a gram-negative bacterium, produces glycoproteins.
  • Utilizing methods known per se we have introduced the C. jejuni gene encoding AcrA, a glycoprotein, into E. coli, This results in the expression of non-glycosylated AcrA protein (see FIG. 2 , step Ib).
  • an operon of C. jejuni encoding a) specific glycosyltransferases and b) an OTase was introduced into E. coli. This resulted in the production of specifically glycosylated AcrA protein according to the invention (see FIG.
  • jejuni recognized the same consensus sequence as the OTase of eukaryotes and archaea and transferred the oligosaccharide by the same proposed mechanism to the protein [Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wren, B. W. and Aebi, M. (2002). N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science, 298: 1790-1793].
  • glycosyl transferases and oligosaccharyl transferases utilized to genetically modify E. coli can be of prokaryotic or eukaryotic origin as glycosyl transferases are ubiquiteous and oligosaccharyl transferases are known from archaea.

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US10/506,917 2002-03-07 2003-03-05 System and method for the production of recombinant glycosylated proteins in a prokaryotic host Abandoned US20050287628A1 (en)

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US12/219,383 US8703471B2 (en) 2002-03-07 2008-07-21 System and method for the production of recombinant glycosylated proteins in a prokaryotic host
US14/189,673 US20140335127A1 (en) 2002-03-07 2014-02-25 System and method for the production of recombinant glycosylated proteins in a prokaryotic host

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US20090074798A1 (en) * 2002-03-07 2009-03-19 Eth Zurich System and method for the production of recombinant glycosylated proteins in a prokaryotic host
US8753864B2 (en) 2005-05-11 2014-06-17 Eth Zurich Recombinant N-glycosylated proteins from procaryotic cells
US8846342B2 (en) 2009-11-19 2014-09-30 Glycovaxyn Ag Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells
US8871491B2 (en) 2010-05-06 2014-10-28 Glycovaxyn Ag Capsular gram-positive bacteria bioconjugate vaccines
US8895014B2 (en) 2008-02-20 2014-11-25 Glycovaxyn Ag Bioconjugates made from recombinant N-glycosylated proteins from procaryotic cells

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CA3125293A1 (en) 2012-11-07 2014-05-15 Glaxosmithkline Biologicals Sa Production of recombinant vaccine in e. coli by enzymatic conjugation
JP6553517B2 (ja) 2013-01-17 2019-07-31 アルサニス・バイオサイエンスズ・ゲゼルシャフト・ミト・ベシュレンクテル・ハフツング Ｍｄｒ大腸菌特異的抗体
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