WO2015013116A1 - Procédé de réduction de l'étendue d'o-mannosylation de glycoprotéines - Google Patents

Procédé de réduction de l'étendue d'o-mannosylation de glycoprotéines Download PDF

Info

Publication number
WO2015013116A1
WO2015013116A1 PCT/US2014/047120 US2014047120W WO2015013116A1 WO 2015013116 A1 WO2015013116 A1 WO 2015013116A1 US 2014047120 W US2014047120 W US 2014047120W WO 2015013116 A1 WO2015013116 A1 WO 2015013116A1
Authority
WO
WIPO (PCT)
Prior art keywords
mannose
seq
lmann
linked
glycoprotein
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.)
Ceased
Application number
PCT/US2014/047120
Other languages
English (en)
Inventor
Stephen R. Hamilton
Sujatha Gomathinayagam
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.)
Organon Pharma UK Ltd
Merck Sharp and Dohme LLC
Original Assignee
Merck Sharp and Dohme Ltd
Merck Sharp and Dohme LLC
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 Merck Sharp and Dohme Ltd, Merck Sharp and Dohme LLC filed Critical Merck Sharp and Dohme Ltd
Publication of WO2015013116A1 publication Critical patent/WO2015013116A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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)
    • C12Y302/01024—Alpha-mannosidase (3.2.1.24)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • 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

Definitions

  • the present invention relates to the field of glycoengineering, and provides methods for removing O-linked mannose residues from intact glycoproteins, including hydrolyzing the Man- a-O-Ser/Thr glycosidic bond.
  • glycoproteins are strongly dependent on the composition of their glycans. Glycosylation is highly dependent on the cellular production system, the particular production clone and the culture process.
  • the methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to secrete large amounts of protein and to perform post-translational modifications, including glycosylation.
  • the nature of the oligosaccharides that are present on a recombinant glycoprotein can impact protein folding, stability, trafficking, functional activity and immunogenicity.
  • N-linked glycosylation patterns differ significantly from those of humans.
  • the modification of N-linked glycans in the golgi typically involves a series of additions of mannose residues by different mannosyltransferases, which results in "outer chain” glycosylations.
  • Such modifications are generally undesired because it can lead to heterogeneity of a recombinant protein product, both from the perspective of molecular weight and composition, thereby complicating protein purification.
  • O-glycosylation is also referred to as O-mannosylation since it is primarily composed of two, three, or four a- 1 ,2-linked mannose (Man) residues, which are attached to an initiating oc-linked mannose residue attached to either a serine or threonine on the protein backbone (Duman et al. 1998).
  • the a- 1 ,2-Man polymers can be further capped by a -1 ,2-Man disaccharide or phosphomannose ( Figure 1 A) (Trimble et al. 2004).
  • the presence of ⁇ -linked mannose on glycans on recombinant proteins is of concern since it has been associated with increased immunogenicity.
  • Man-a-O-Ser/Tlir glycosidic bond on intact glycoproteins secreted from glycoengineered P. pastoris would enhance the value and the utility of yeast expression systems in general, and P. pastoris expression systems in particular, for the production of therapeutic glycoproteins.
  • the invention provides methods to reduce the extent of O-mannosylation on intact glycoproteins.
  • the extent of O-mannosylation is reduced on intact glycoproteins produced in yeast or fungal host cells.
  • the invention also provides methods to hydrolyze the Man-a- ⁇ 9-Ser/Thr glycosidic bond on intact glycoproteins produced in yeast or fungal host cells.
  • glycoproteins including proteins expressed in glycoengineered P. pastoris.
  • the disclosed invention provides a strategy to modify the O-linked glycosylation pattern or O- glycoprofile of recombinant proteins that possess O-linked mannose residues, exemplified by proteins that are expressed in P. pastoris and S. cerevisiae.
  • the disclosed methods can be used to facilitate the removal of O-mannosylation from therapeutic proteins produced in any expression system that is characterized by an O-glycoprofile that adds O-linked alpha-mannose residues.
  • the invention provides a method for reducing the extent of O- mannosylation of a recombinant glycoprotein comprising the steps of contacting a secreted recombinant glycoprotein comprising O-linked mannose residues with lysosomal mannosidase under conditions which allow for the enzymatic activity of mannosidase to produce a
  • glycoprotein composition which comprises a reduced level of O-mannosylation compared to the O-mannosylation of the recombinant glycoprotein that is not treated with lysosomal
  • mannosidase In a particular embodiment, at least one a 1 ,2 mannose, a 1 ,3 mannose and/or a 1 ,6 mannose residue in an O-linked mannose polymer is hydrolyzed. In an alternative embodiment the disclosed methods remove O-mannose residues from intact glycoproteins by hydrolyzing the Man-a-O-Ser/Thr glycosidic bond.
  • the methods of the invention can be practiced using a host cell capable of producing the Man-a-O-Ser/Thr glycosidic bond, such as, but not limited to yeast or fungal host cells.
  • a host cell capable of producing the Man-a-O-Ser/Thr glycosidic bond such as, but not limited to yeast or fungal host cells.
  • the methods of the invention can be practiced using wild-type S.cerevisiae or glycoengineered Pichia pastoris host cells.
  • Suitable host cells include Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia mem.br anaefaciens, Pichia opuntiae, Pichia thermotolerans , Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum and Neurospora crassa.
  • the methods of the invention can be practiced using a therapeutic glycoprotein selected from an antibody, a hormone, a cytokine, an enzyme or a bioactive peptide, which is produced in an expression system which performs O-glycosylation in a manner which produces single O-linked mannose or oligomannosyl-glycans that differ from the mucin-type glycans produced by human cells.
  • a therapeutic glycoprotein selected from an antibody, a hormone, a cytokine, an enzyme or a bioactive peptide, which is produced in an expression system which performs O-glycosylation in a manner which produces single O-linked mannose or oligomannosyl-glycans that differ from the mucin-type glycans produced by human cells.
  • Heterologous protein products having reduced O- glycosylation produced using the disclosed methods are also part of the present invention.
  • the treated glycoprotein is optionally isolated/purified and its O-glyoprofile is characterized using an appropriate analytical protocol (i.e., high-performance anion exchange chromatorgraphy with pulsed amperometric detection (HPAEC-PAD or quadruple time of flight (Q-TOF) mass spectrometry).
  • HPAEC-PAD high-performance anion exchange chromatorgraphy with pulsed amperometric detection
  • Q-TOF quadruple time of flight
  • a functional assay, or a PK/PD determination can be performed to evaluate the effect of the reduced level of O-mannosylation on the activity profile or half-life of the treated glycoprotein.
  • the methods can be practiced on any recombinant glycoprotein possessing at least one
  • the method can be used to modify the O-glycoprofile of recombinant glycoprotein possessing an O- linked glycan comprising a mannose residue directly linked to a serine or threonine residue of the glycoprotein by an ot-bond.
  • recombinant glycoprotein characterized by an O- linked glycan comprising mannose polymers, wherein at least one al ,2 mannose, al ,3 mannose and/or al ,6 mannose is linked to the initiating serine or threonine linked mannose residue can be modified using the disclosed methods.
  • the disclosed methods can be used to enzymatically remove at least 10% of the original O-linked mannose present on the recombinant glycoprotein. In some embodiments, depending on the reporter protein and the reaction conditions, greater than 74% of the O-linked mannose is removed. Alternatively, the methods can be used to enzymatically cleave at least 10% of the Man- ⁇ -0-Ser/Thr glycosidic bonds present on an O-glycosylated glycoprotein. In particular embodiments, greater than 74%> of the Man-a-O-Ser/Thr glycosidic bonds are cleaved.
  • the disclosed methods can be used to remove at least 50 %, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, or at least about 99% of the O-linked mannose residues present on the glycoprotein.
  • the disclosed methods can be used to enzymatically cleave at least 50 %, 60%, 70%, 80%, 85%, 90%), 95%), 97.5%), or at least about 99%> of the Man-a-O-Ser/Thr glycosidic bonds present on an O-glycosylated glycoprotein.
  • the disclosed methods can be used to completely remove all (e.g, 100% reduction) of the mannose residues and/or all of the Man-a-O-Ser/Thr glycosidic bonds present on an O-glycosylated protein of interest.
  • the methods of the invention can be practiced in vitro or in vivo using a recombinant lysosomal mannosidase (LMann) selected from the enzyme class consisting of E.C.3.2.1.24.
  • LMann lysosomal mannosidase
  • Suitable lysosomal mannosidases can be isolated from a eukaryotic source.
  • the methods of the invention can be practiced using a LMann isolated from a source organism selected from Arabidopsis thaliana, Dictyostelium discoideum, Glycine max, Cavia porcellus, Homo sapiens, Medicago truncatula, Mus musculus, Ricinus communis, Sulfolobus solfataricus, Trypanosoma cruzi, Solanum lycopersicum, or Vitis vinifera.
  • a source organism selected from Arabidopsis thaliana, Dictyostelium discoideum, Glycine max, Cavia porcellus, Homo sapiens, Medicago truncatula, Mus musculus, Ricinus communis, Sulfolobus solfataricus, Trypanosoma cruzi, Solanum lycopersicum, or Vitis vinifera.
  • the methods of the invention can be practiced using LMann isolated from a plant or non-plant eukaryotic source. As shown herein the disclosed methods can be practiced using a lysosomal mannosidase selected from Glycine max (GmMann), Medicago truncatula (MtLMann) and Vitis vinifera (VvLMann) or a lysosomal mannosidase selected from Glycine max (GmMann), Medicago truncatula (MtLMann) and Vitis vinifera (VvLMann) or a lysosomal mannosidase selected from Glycine max (GmMann), Medicago truncatula (MtLMann) and Vitis vinifera (VvLMann) or a lysosomal mannosidase selected from Glycine max (GmMann), Medicago truncatula (MtLMann) and Vitis
  • Arabidopsis thaliana Arabidopsis thaliana (AtLMann) and Homosapiens (HsLMann).
  • the disclosed methods can be practiced using a lysosomal mannosidase obtained from another eukaryotic source.
  • a LMann obtained from a eukaryotic cell can be expressed, purified and screened using the methods disclosed in this invention.
  • the methods and Examples demonstrate how a LMann can be recombinantly expressed in P. pastoris, isolated and then used in vitro to reduce O-mannosylation of a glycoprotein possessing O-linked mannose residues.
  • Another approach is to co-secrete the LMann from the same cell as that expressing the glycoprotein of interest.
  • the methods of the invention can be practiced using a LMann selected from, but not limited to homologues of the human lysosomal mannosidase (HsLMann) enzyme (AAC34130).
  • HsLMann human lysosomal mannosidase
  • the methods of the invention can be practiced using a LMann selected from the mannosidases described using the designations provided in Table 1 to denote the LMann sequences having the amino acid sequences disclosed herein, including: AtLMann (SEQ ID NO: 1), DdLMann (SEQ ID NO: 2), GmMann (SEQ ID NO: 3), GpLMann (SEQ ID NO: 4), HsLMann (SEQ ID NO: 5), MtLMann (SEQ ID NO: 6), MmLMann (SEQ ID NO: 7), RcLMann (SEQ ID NO: 8), SsLMann (SEQ ID NO: 9),TcLMann (SEQ ID NO:
  • reaction conditions including buffer composition, pH, time of incubation, temperature, substrate conentration, enzyme concentration, etc.
  • reaction conditions including buffer composition, pH, time of incubation, temperature, substrate conentration, enzyme concentration, etc.
  • reaction conditions described in the invention can be optimized to further enhance O-linked mannose removal.
  • a person having ordinaiy skill could readily use the reaction conditions described in the invention, or optimized conditions to scale the disclosed methods to faciliate O-mannose reduction of commercial scale preparations of therapetuic O- mannosylated glycoproteins.
  • lysosomal a-mannosidases are large proteins, typically comprising a heterodimer of around 150kDa, which later self-dimerizes.
  • the heterodimers are cleavage products of cleaved single chain precursor peptides. It is this latter single chain precursor that has been used to exemplify the utility of LMann to remove O-linked mannose. It is foreseeable that a cleaved, truncated or subdomain variant of the enzyme may be more efficient at removing O- linked mannose on particular proteins due to their differing structural conformations.
  • the methods described herein can be practiced using a subdomain variant of a recombinant LMann.
  • artificial internal protease cleavage sites have been added to facilitate processing of the single chain precursor peptide into the heterodimeric form.
  • the methods of the invention can be practiced using an N- or C-terminal variant of one of the recombinant lysosomal
  • the methods of the invention can be practiced ex vivo (e.g., in vitro using a glycoprotein that is secreted by a host cell), by the exogenous addition of a lysosomal mannosidase to a glycoprotein of interest following expression and secretion and/or optionally purification of the glycoprotein.
  • the glycoprotein can remain in the culture media or can be purified.
  • the methods of the invention are practiced in vivo using yeast production strains that are engineered to co-secrete a lysosomal mannosidase in the presence of the glycoprotein of interest under conditions which promote the removal of O-linked mannose residues and/or the hydrolysis of the Man-a-0-Ser/Thr glycosidic bond during secretion or after secretion from the host cell.
  • the methods of the invention can be practiced in vivo.
  • one of the LMann expression vectors described herein could be used to directly transform a yeast production strain that is already engineered to express the protein of interest.
  • Figure 1 Illustrative representation of native and engineered O-linked glycosylations.
  • A Comparison of mammalian mucin- and oc-dystroglycan-type O-glycosylations to wild-type P. pastoris O-glycosylation.
  • B A summary of in vivo engineering and in vitro manipulations to reduce O-mannosylation on proteins secreted from glycoengineered P. pastoris.
  • FIG. 1 Exemplification of an expression vector used to produce recombinant lysosomal mannosidase.
  • the vector map for pGLY12387 represents the expression vector used to express the VvLMann under the control the AOXl promoter. This vector integrates at the TRP2 loci of the P. pastoris genome and uses the Zeocin resistance marker for selection. Restriction enzyme sites used for subcloning or linearization (underlined) of the vector for transformation are highlighted.
  • B The open reading frame encoding the VvLMann possesses an N-terminal a- mating factor secretion signal (SEQ ID NO: 13) fused in frame to a double FLAG tag, the sequence of which is in the upper box.
  • SEQ ID NO: 13 N-terminal a- mating factor secretion signal
  • FIG. 3 Preliminary screening to identify potential O-glycan cleavage ability of LMann secreted from P. pastoris.
  • the supernatant from P. pastoris transformed with vectors encoding twelve homologues of lysosomal mannosidase (see Table 1) were assessed for their ability to reduce the O-glycan profile of recombinant SCI-CTP produced in a GSOl .0 glycoengineered P. pastoris strain.
  • the SCI-CTP glycoprotein profile was analyzed by Q-TOF (A) and demonstrated to possess between six and twelve mannose residues per molecule.
  • FIG. 4 Preliminary characterization of VvLMann.
  • Panels A and B represent untreated control SCI-CTP or that incubated for 24h with supernatant from P. pastoris transformed with the VvLMann expression vector.
  • C O-mannose profile
  • Incubation of SCI-CTP with a-l ,2-mannosidase from Trichodema reesei (TrMannI) in the presence or absence of VvLMann are respresented in (D) and (E) respectively.
  • the black arrows highlight peaks corresponding to the unglycosylated mass (8726 amu) of SCI-CTP.
  • FIG. 1 Purification of LMann. Recombinant VvLMann GmLMann and MtLMann were purified by nickel column chromatography from P. pastoris supernatants. Aliquots of the purified enzymes were run non-reduced (NR) or reduced (R) on SDS-PAGE gels and detected by Coomassie blue staining (A) or immunoblotted with anti-HIS tag antibody (B). Molecular weights markers are included for confirmation of molecular weight.
  • NR non-reduced
  • R reduced
  • A Coomassie blue staining
  • B immunoblotted with anti-HIS tag antibody
  • FIG. 6 Characterization of the ability of purified LMann to reduce the O-glycan profile of SCI-CTP.
  • Aliquots of VvLMann, GmLMann or MtLMann incubated with SCI-CTP for 24h produced the Q-TOF profiles represented in panels B, C, and D respectively.
  • Untreated control SCI-CTP incubated for 24h in the absence of LMann is represented in panel A.
  • the black arrows highlight peaks corresponding to the unglycosylated mass (8726 amu) of SCI-CTP.
  • FIG. 7 Characterization of the ability of purified LMann mannosidases, which did not show activity when screened as supernatant, to reduce the O-glycan profile of SCI-CTP.
  • Incubation of AtLMann and HsLMann with SCI-CTP for 24h produced the Q-TOF profiles represented in panel (B) and (C) respectively.
  • Untreated control SCI-CTP incubated for 24h in the absence of LMann is represented in panel (A).
  • the black arrows highlight peaks corresponding to the unglycosylated mass (8726 amu) of SCI-CTP.
  • Figure 8 Purification of AtLMann and HsLMann.
  • AtLMann and HsLMann were purified by nickel column chromatography from P. pastoris supernatants. Aliquots of the purified enzyme were run reduced on SDS-PAGE gels and detected by Coomassie blue staining (A) or immunoblotted with anti-HIS tag antibody (B). Molecular weights markers are included for confirmation of molecular weight.
  • FIG. 9 Characterization of VvLMann enzymatic activity.
  • Q-TOF analysis of SCI-CTP incubated for 24h in the absence of LMann is represented in panel A.
  • Q-TOF analyses of parallel reactions following the addition of VvLMann to SCI-CTP in the presence of ImM zinc (B), no additional zinc (C) or ImM EDTA (D) are represented.
  • the black arrows highlight peaks corresponding to the unglycosylated mass (8726 amu) of SCI-CTP.
  • FigurelO Characterization of the ability of purified VvLMann to reduce the O-glycan profile of human FcDM SEQ1.
  • the black arrows highlight peaks corresponding to the unglycosylated mass (50393+/-1 amu) of hFcDM SEQ1.
  • FIG. 11 Characterization of the ability of purified VvLMann to reduce the O-glycan profile of SCI-OPEP. SCI-OPEP incubated overnight in the absence (A) or presence (B) of purified VvLMann was assessed by Q-TOF. The black arrow highlights the peak corresponding to the unglycosylated mass (7869 amu) of SCI-OPEP.
  • Figure 12 Characterization of the ability of purified VvLMann to reduce the O-glycan profile of commercial Leukine ® .
  • An aliquot of commercial Leukine ® was PNGase treated to remove N- linked glycosylation. Subsequently this sample was incubated overnight in the absence (A) or presence (B) of VvLMann and assessed by Q-TOF.
  • the black arrow highlights the peak corresponding to the unglycosylated mass (14431 amu) of Leukine ® .
  • Phosphomannose containing O-linked glycoforms have been labeled with a "P".
  • Figure 13 Production of recombinant LMann subdomains.
  • Lysosomal mannosidase is typically synthesized as a precursor peptide which is processed into N- and C-terminal domains.
  • An alignment (A) of the human LMann sequence with that of Vv-, GM- and Mt-LMann depicts where the human enzyme is cleaved (black arrow) into the two separate subdomains, based on the characterization by Berg et al. (2001) Mol. Gen. Met. 73, 18-29.
  • Panel B depicts a variety of forms of secreted recombinant VvLMann.
  • Form I represents the intact secreted precursor of the VvLMann (regular hatched box) possessing an N-terminal double FLAG tag (black box) and a C-terminal HIS tag (white box).
  • the black arrow depicts where the VvLMann would be proteolytically processed into two subdomains, based on homology to the HsLMann.
  • Form II represents the introduction of a Kex2p cleavage (bolded hatched box) site into the VvLMann precursor sequence to facilitate N- and C-terminal processing.
  • Form III represents Form II with the addition of a HIS tag N-terminal to the Kex2p site to facilitate purification.
  • Form IV is similar to Form II except that a factor Xa site (FXa, dotted box) is introduced in place of the Kex2p site, thus allowing cleavage post-secretion.
  • Form V represents the individual expression of the VvLMann N-terminal domain with an N-terminal double FLAG tag and a C-terminal HIS tag.
  • Form VI represents individual expression of the VvLMann C-terminal domain with an N- terminal double FLAG tag and a C-terminal HIS tag.
  • the present invention is based on the discovery that lysosomal a-mannosidases, can reduce the extent of O-mannosylation on intact glycoproteins and hydrolyze the Man-a-O- Ser/Thr glycosidic bond of glycoproteins, particularly those produced in wild type and engineered yeast expression systems.
  • the disclosed lysosomal a-mannosidases are efficient at O-mannose removal and because they are recombinant they provide purified mannosidase preparations that are devoid of the undesired proteolytic activity associated with crude cell extracts.
  • Using the disclosed lysomsomal mannosidases in the methods disclosed and claimed herein provides a scaleable strategy for the removal of O-linked glycosylation from glycoproteins expressed in P. pastoris, or any other expression system characterized by O- mannosylation.
  • the disclosed invention provides a means to produce biotherapeutic glycoproteins in non-mammalian expression systems that are more homologous to glycoproteins produced in mammalian expression systems.
  • P. pastoris like other yeast, add O-linked oligosaccharides to the hydroxyl groups of serine and threonine residues of secreted proteins.
  • O-linked glycan structures are typically polymers of between 1 and 4 a-l ,2-linked mannose residues, with a subset of glycans being potentially capped by a ⁇ - 1 ,2-Man disaccharide or phosphomannose.
  • Such O-mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses.
  • the recombinant proteins expressed in this system are devoid of phosphomamiose and B-mannose on O-linked glycans, resulting only in a-l ,2-mannose polymers (Hopkins et al. 201 1).
  • O-glycosylated recombinant proteins expressed in P. pastoris are well documented in the literature (Duman et al. 1998; Bewley et al. 1999; Letourneur et al. 2001 ; Boraston et al. 2003; Trimble et al. 2004; O'Leary et al. 2004).
  • extended sialylated O-linked glycosylation is typically of the mucin- or a- dystroglycan-type O-glycosylation (Hanisch 2001 ; Lommel and Strahl 2009).
  • O-glycosylation is often referred to as O- mannosylation since it is primarily composed of one, two, or three a-l ,2-linked mannose (Man) residues, which are attached to an initiating a-linked mannose residue attached to either a serine or threonine on the protein backbone (Duman et al. 1998).
  • Man mannose
  • the a- 1 ,2-Man polymers can be additionally capped by a ⁇ -l ,2-Man disaccharide or phosphomannose ( Figure 1 A) (Trimble et al. 2004).
  • An alternative approach is to modulate the activity of the protein O-mannosyltransferase (PMT) family of genes, which are responsible for the addition of the initiating mannose to the serine or threonine residues of the protein backbone.
  • PMT protein O-mannosyltransferase
  • the knockout of individual PMT genes can cause severe growth defects in yeast (Gentzsch and Tanner 1996; Gentzsch and Tanner 1997). As such, disrupting the entire repertoire of PMT genes is considered to be difficult, with any resultant strains being potentially non-viable (Hopkins et al. 201 1).
  • An alternative approach is to suppress Pmtp activity by adding chemical inhibitors (PMTi) to the fermentation media during cultivation ((Orchard et al. 2004). However this strategy only reduces the occupancy of O-linked glycans, and does not completely eliminate it.
  • Enzymatic demannosylation provides yet another option for reducing the extent of O- mannosylation of glycoproteins produced in Pichia production strains.
  • Jack bean a-l,2/3/6-mannosidase has been shown to remove O-linked mannose from proteins expressed in wild type yeast.
  • Bergwerff demonstrated that Jack bean mannosidase could remove O-linked mannose fromrecombinant leech-derived tryptase inliibitor (rLDTI), following treatment will alkaline phosphatase to remove phosphomannose residues (Bergwerff et al. 1998).
  • rLDTI leech-derived tryptase inliibitor
  • the reported ability of Jack bean a-mannosidase activity to remove O-linked mannose is primarily restricted to relatively small glycopeptides.
  • Jack bean mannosidase does not provide a universal option for the removal of O- mannosylation, because the enzymatic activity is believed to be limited by the conformation of the particular target protein and there is an inherent resistance of the Man-O-Ser/Thr bond to hydrolysis.
  • Another potential limitation on the utility of Jack bean mannosidase as an option for reducing the extent of O-mannosylation on therapeutic glycoproteins is that the only current source of Jack bean mannosidase is from crude cell extracts. As such, these possess numerous proteases which can degrade the therapeutic glycoprotein of interest during the deglycosylation incubation.
  • WO2005/033325 purports to describe a method of producing recombinant polypeptides in fungal host cells that are free of O-linked mannose residues using either a commercially available Jack bean a-mannosidase preparation or recombinant Thermotoga maritima a- mannosidase (TMM) expressed in E.coli. Activities of the Jack bean mannosidase and the TMM were shown using two different relatively simple reporter proteins (tetra-glycosylated human GLP-1 and mono-glycosylated human insulin MG2). The results produced using Jack bean mannosidase agreed with what had been previously published by others, including Bergwerff et al. and Ibatullin et al.
  • TMM of the GLPl While the demannosylation by TMM of the GLPl was efficient at 40°C, elevated temperatures (70°C) were required for the human insulin demannosylation. Such elevated temperatures are not desirable when handling therapeutic glycoproteins on a commercial scale since it may lead to increased aggregation, proteolysis and/or denaturation of the therapeutic protein. Furthermore, the methods provided for the TMM, indicate that the incubation conditions are protein specific and require optimization for each protein demannosylated, specifically optimization of the incubation temperature.
  • maritima mannosidase was so low towards the human LMann used in the Blastp search described below, that it was not returned as a homologue in the BLAST search that identified the eleven homologues used to exemplify the current invention.
  • both the T. maritima mannosidase and the SsLMann are considered to be significantly different to the eurkaryotic lysosomal mannosidases, which are the primary focus of the current invention, and represent a different family of enzymes.
  • the Sulfolobus solfataricus protein was named SsLMann and characterized in parallel to the eukaryotic LManns, though this former enzyme is clearly not a lysosomal mannosidase.
  • Jack bean mannosidase has not been reported to date in public databases, and commercial preparations are prepared from crude cellular extracts, and not a recombinant enzyme. As such, it is possible that non-desirable cellular components may be present, including proteases which may hydrolyze the glycoprotein of interest. Accordingly, the use of Jack bean mannosidase in a production scale process is not desirable from the perspective of sourcing, with the possibility of contaminating protease activities, reagent quality, and cost. Definitions
  • Lysosomal mannosidase refers to a eukaiyotic mannosidase that localizes to the lysosome of a eukaiyotic cell and possesses a-mannosidase activity.
  • Lysosomal mannosidases are members of the glycoside hydrolase family 38 (GH38, Class II). Lysosomal mannosidases catalyze the hydrolysis of ⁇ -1 ,2-, ⁇ -1 ,3- and a-l,6-glycosidic bonds with retention of configuration of the anomeric carbon of the released mannose residue.
  • lysosomal mannosidase lysosomal alpha-mannosidase
  • mannosidase alpha-B mannosidase (alpha B) lysosomal
  • lysosomal acid alpha-mannosidase lysosomal acid alpha-mannosidase
  • O-mannosylation as it relates to the covalent conjugation of one or more mannose residues to serine or threonine residues of a protein sequence.
  • O-glycoprofile refers to the O-linked glycosylation profile of a released O-linked glycan population or the profile of the said O-linked glycan population while conjugated to a peptide sequence.
  • Man-a-O-Ser/Thr glycosidic bond refers to the glycosidic bond connecting a mannose residue to a serine or threonine residue in a peptide sequence, where the mannose residue is in the ct-configuration.
  • the term “Man-a-O-Ser/Thr glycosidic bond” is also referred to as “Man-O-Ser/Thr glycosidic bond” or “Man-O-Ser/Thr O-glycosidic bond", all meaning the same defined glycosidic bond structure.
  • the term "intact” refers to a glycoprotein that has not been manipulated in vitro to alter the conformation or structure of the protein.
  • manipulation include, but are not limited to, denaturization of the protein by physical or chemical means and/or degradation of the protein by physical, chemical or enzymatic means.
  • Typical manipulations used in the art are heat denaturization through increased temperature, proteolytic degradation using proteolytic enzymes and chemical denaturization using detergents or reducing agents. Lysosomal Mannosidases
  • a-Mannosidases are ubiquitous in nature and have been purified and characterized from various plant, microbial and animal sources. a-Mannosidases have been classified into two independently derived groups, class I and class II, based on biochemical properties, substrate specificity, inhibitor profiles and sequence alignments. Generally speaking, Class I contains mannosidases found in the endoplasmic reticulum (ER) and Golgi. In eukaiyotes, class I a- mannosidases are involved in early N-glycan processing reactions and in N-glycan dependent quality control in the ER. Class I a-mannosidases are conserved throughout eukaryotic evolution and do not share sequence homology with class II a-mannosidases.
  • the second group (class II) is more heterogeneous and contains the lysosomal mannosidases and other distantly related enzymes.
  • a class of lysosomal mannosidases is more heterogeneous and contains the lysosomal mannosidases and other distantly related enzymes.
  • LManns with broad substrate specificity and the ability to catalyze the hydrolysis of a- 1,2, a- 1 ,3 and a-l ,6-glycosidic bonds reside in the lysosome of eukaryotic cells and function to degrade mannose containing glycans.
  • LManns (EC 3.2.1.24) (designated lysosomal a-1,2/3/6- mannosidases), are members of the glycoside hydrolase family 38 (GH38, Class II). They are involved in the catabolism of Asn-linked glycans and play a vital role in maintaining cellular homeostasis.
  • lysosomal mannosidases results in the lysosomal storage disease a-mannosidosis (Malm and Nilssen 2008). Patients having this disease display a wide range of neurological, immunological and skeletal symptoms caused by a multisystemic accumulation of mannose containing oligosaccharides (Malm and Nilssen 2008). As such, much work has been done to characterize and understand this enzyme (Berg et al. 2001). Much of this work has focused on characterizing the enzyme in terms of its activity towards released free N-linked glycofonns (Aronson, Jr. and Kuranda 1989). As a consequence, lysosomal mannosidase has been shown to have a similar activity towards free N-linked glycans as Jack bean a-1 , 2/3/6- mannosidase.
  • HsLMann human lysosomal mannosidase
  • AAC34130 The entire human lysosomal mannosidase sequence (AAC34130) was used to BLAST search NCBI databases for homologues. Either the BLAST or the BLAST 2.0 algorithms, described in Altschul et al. (1977) Nucl. Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively could be used to search for homologues. Specifically the NCBI Standard Protein BLAST website:
  • solfataricus showed relatively low homology (approxiamtely 1 1 % homology), and was the only non-eukaiyotic protein assessed in the current invention.
  • Sulfolobus solfataricus protein was named SsLMann and characterized in parallel to the eukaryotic LManns, though this fonner enzyme is clearly not a true lysosomal mannosidase. It is foreseeable that other homologues identified in the described Blastp search, or from an expanded search, can be selected and screened using the methods disclosed for their ability to
  • LMann homologues in a strains engineered to produce N-linked glycans possessing Man5GlcNAc2 glycoforms, designated GS2.0 glycans, could be more active due to reduced hyperglycosylation of the N-linked glycans.
  • producing the LMann with complex-type N-linked glycans may be more beneficial.
  • the treated glycoprotein is optionally isolated/purified and its O- glycoprofile is characterized using an appropriate analytical protocol (e.g., high-performance anion exchange chromatorgraphy with pulsed amperometric detection (HPAEC-PAD or quadrople time-of-flight (Q-TOF) mass spectrometry of the glycopeptide or a parallel mass spectrometry based technology to analyze either the O-glycan composition or O-glycoprofile of a peptide/protein; gel-shift assays can be used to monitor overall peptide size and changes in mass following deglycosylation using the methods disclosed; likewise capillary electrophoresis may be used to monitor changes in the mass of a glycopeptide or glycan structure, for instance using a microchip such as that used in the LabChip technology; nuclear magnetic resonance (NMR), or a similar structural determination technique, can be used to monitor the structure of released O- glycans or glycopeptides; a functional assay can be used to evaluate the effect of
  • the treated glycoprotein is a glycoprotein produced in a wild-type or engineered yeast or fungal cell selected from SCI-CTP, SCI-OPEP, human Fc, GM-CSF or another protein which has been previously demonstrated to be O-glycosylated.
  • O-glycosylated therapeutic proteins are reviewed by Higgins (Higgins 2010).
  • Other potential proteins that can benefit from the methods disclosed are those which are traditionally not O- glycosylated in their native state but which are when expressed in a heterologous system, such as in yeast or fungal systems.
  • the methods described herein can be used to remove O- linked mannose on proteins from mammalian or higher eukaryotic sources.
  • IgG2 produced in CHO and COS cell lines has been reported to possess a single O-linked mannose on the light chain (Martinez et al. 2007).
  • the methods disclosed herein can be used in combination with other glycosidic enzymes to remove oc-dystroglycan-type glycans on proteins expressed in higher eukaryotes. In such instances, this can not only be used in the production of recombinat therapeutic proteins, but also be used to improve the analytical characterization of these proteins, which may be restricted by the presence of O-linked glycan structures.
  • the present invention encompasses any isolated Pichia sp. host cell (e.g. , such as Pichia pastoris) including wild-type and engineered host cells comprising various modified constructs, including host cells that comprise a promoter e.g., operably linked to a polynucleotide encoding a heterologous polypeptide ⁇ e.g. , a reporter or immunoglobulin heavy and/or light chain) as well as methods of use thereof, e.g., methods for expressing the heterologous polypeptide in the host cell.
  • Host cells of the present invention may be also genetically engineered so as to express particular glycosylation patterns on polypeptides that are expressed in such cells.
  • Host cells of the present invention are discussed in detail herein. Any engineered Pichia host cell cultured under any of the described conditions forms part of the present invention.
  • the host cell is selected from the group consisting of any Pichia cell, such as
  • Pichia pastoris Pichia angusta (Hansenula polymorpha), Pichia flnlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens , Pichia minuta ⁇ Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans , Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia.
  • Pichia host cells ⁇ e.g., Pichia pastoris
  • Pichia pastoris can be genetically engineered to eliminate glycoproteins having a-mannosidase-resistant N-glycans by deleting or disrupting one or more of the beta-mannosyltransferase genes (e.g., BMTl, BMT2, BMT3, and BMT4) (See, U.S. Patent No. 7,465,577) or abrogating translation of RNAs encoding one or more of the beta-mannosyltransferases using interfering RNA, antisense RNA, or the like.
  • the beta-mannosyltransferase genes e.g., BMTl, BMT2, BMT3, and BMT4
  • the scope of the present invention includes the use of such engineered Pichia host cells (e.g., Pichia pastoris) comprising an expression cassette ⁇ e.g., a promoter operably linked to a heterologous polynucleotide encoding a heterologous polypeptide).
  • Pichia host cells e.g., Pichia pastoris
  • an expression cassette e.g., a promoter operably linked to a heterologous polynucleotide encoding a heterologous polypeptide.
  • Engineered host cells ⁇ e.g., Pichia pastoris) cultured under conditions of the present invention also include those that are genetically engineered to eliminate glycoproteins having phosphomannose residues, e.g., by deleting or disrupting one or both of the phosphomannosyl transferase genes PNOl and MNN4B (See for example, U.S. Patent Nos. 7,198,921 and
  • an engineered Pichia host cell has been genetically modified to produce glycoproteins that have predominantly an N-glycan selected from the group consisting of complex N-glycans, hybrid N-glycans, and high mannose N-glycans wherein complex N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 3 GlcNAc 2 , GlcNAc (
  • N-glycans are, in an embodiment of the invention, selected from the group consisting of GlcNAcMan(3-5 ) GlcNAc 2 , GalGlcNAcMan(3_ 5 ) GlcNAc 2 , and NANAGalGlcNAcMan ( 3_ 5) GlcNAc 2 ; and high mannose N-glycans are, in an embodiment of the invention, selected from the group consisting of Man 5 GlcNAc 2 ,
  • an engineered Pichia host cell has been genetically modified to produce glycoproteins that have predominantly an O-glycan selected from the group consisting of wild-type or engineered O-glycans wherein wild-type glycans are inititated by an a-linked mannose residue to serine or threonine of a peptide sequence, which may subsequently be extended by 1 -5 mannose residues in an a- or ⁇ -linked configuration and can possess phosphate linked to particular mannose residues.
  • Engineered O-glycans are composed of those where the extent of ⁇ -, ⁇ - or phospho-mannose have been reduced and can further result in the production of O- linked glycans where single mannose residues are conjugated to serine or threonine residues of the peptide backbone.
  • the scope of the present invention includes such engineered Pichia host cells (e.g., Pichia pastoris) comprising a modified, truncated, or deleted form of the XRN1 gene.
  • Pichia host cells e.g., Pichia pastoris
  • modified, truncated, or deleted form of the XRN1 gene e.g., Pichia pastoris
  • Additional embodiments of the present invention include engineered Pichia host cells ⁇ e.g., Pichia pastoris) cultured under conditions of the present invention that are genetically engineered to include a nucleic acid that encodes the Leishmania sp. single-subunit
  • engineered host cells e.g., Pichia pastoris
  • engineered host cells also include those that are genetically engineered to eliminate nucleic acids encoding Dolichol-P-Man dependent alpha(l-3) mannosyltransferase, i.e.., Alg3, such as described in US Patent Publication No. US2005/0170452.
  • yeast or filamentous fungus host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.
  • Additional embodiments include the use of other eukaryotic organisms including higher eukaryotics organism, such as insect or mammalian cells for the production of therapeutic proteins, and using methods similar to those described herein to removal O-linked mannose residues.
  • the O-linked mannose structures may be a-dystroglycan-type in nature, which have been reduced to single O-linked mannose residues through genetic engineering of the host cell or through the prior/combined digestion of the a-dystroglycan-type glycans with other glycosidic enzymes, including but not limited to sialidase, galactosidase and hexosaminidase.
  • a “polynucleotide”, “nucleic acid” includes DNA and RNA in single stranded form, double-stranded form or otherwise.
  • a "polynucleotide sequence” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in a nucleic acid, such as DNA or RNA, and means a series of two or more nucleotides. Any polynucleotide comprising a nucleotide sequence set forth herein ⁇ e.g. , promoters of the present invention) forms part of the present invention.
  • a "coding sequence” or a sequence “encoding” an expression product, such as an RNA or polypeptide is a nucleotide sequence ⁇ e.g., heterologous polynucleotide) that, when expressed, results in production of the product (e.g., a heterologous polypeptide such as an immunoglobulin heavy chain and/or light chain).
  • oligonucleotide refers to a nucleic acid, generally of no more than about 100 nucleotides [e.g., 30, 40, 50, 60, 70, 80, or 90), that may be hybridizable to a polynucleotide molecule. Oligonucleotides can be labeled, e.g., by incorporation of 32 P- nucleotides, 3 H-nucleotides, 14 C-nucleotides, 35 S-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • a label such as biotin
  • a “protein”, “peptide” or “polypeptide” includes a contiguous string of two or more amino acids.
  • a “protein sequence”, “peptide sequence” or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide or polypeptide.
  • isolated polynucleotide or “isolated polypeptide” includes a polynucleotide or polypeptide, respectively, which is partially or fully separated from other components that are normally found in cells or in recombinant DNA expression systems or any other contaminant. These components include, but are not limited to, cell membranes, cell walls, ribosomes, polymerases, serum components and extraneous genomic sequences.
  • the scope of the present invention includes the isolated polynucleotides set forth herein, e.g., the promoters set forth herein; and methods related thereto, e.g., as discussed herein.
  • An isolated polynucleotide or polypeptide will, preferably, be an essentially
  • PCR polymerase chain reaction
  • a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g. , directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence to which it operably links.
  • a coding sequence (e.g., of a heterologous polynucleotide, e.g., reporter gene or immunoglobulin heavy and/or light chain) is "operably linked to", "under the control of, “functionally associated with” or “operably associated with” a transcriptional and translational control sequence (e.g., a promoter of the present invention) when the sequence directs RNA polymerase mediated transcription of the coding sequence into RNA, preferably mRNA, which then may be RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
  • RNA preferably mRNA
  • the present invention includes vectors or cassettes which comprise various modified constructs, including promoters optionally operably linked to a heterologous polynucleotide.
  • the term "vector” includes a vehicle (e.g. , a plasmid) by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence.
  • a vector for use herein include plasmids, integratable DNA fragments, and other vehicles that may facilitate introduction of the nucleic acids into the genome of a host cell (e.g., Pichia pastoris).
  • Plasmids are the most commonly used form of vector but all other forms of vectors which serve a similar function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels, et al, Cloning Vectors: A Laboratory Manual, 1985 and Supplements, Elsevier, N.Y., and Rodriguez et al. ( eds.). Vectors: A Survey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, MA.
  • a polynucleotide (e.g., a heterologous polynucleotide, e.g., encoding an immunoglobulin heavy chain and/or light chain), operably linked to a promoter, may be expressed in an expression system.
  • expression system means a host cell and compatible vector which, under suitable conditions, can express a protein or nucleic acid which is carried by the vector and introduced to the host cell.
  • Common expression systems include fungal host cells (e.g., Pichia pastoris) and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • BLAST ALGORITHMS Altschul, S.F., et al., J. Mol. Biol. (1990) 215:403-410; Gish, W., et al. , Nature Genet. (1993) 3 :266-272; Madden, T.L., et al., Meth. Enzymol. (1996) 266: 131-141 ; Altschul, S.F., et al , Nucleic Acids Res. (1997) 25:3389-3402; Zhang, J., et al , Genome Res. (1997) 7:649-656; Wootton, J.C., et al , Comput. Chem. (1993) 17: 149-163;
  • the a-l ,2-mannosidase was cloned from Trichoderma reesei (TrMannl), a C-terminal 6xHIS tag was added and it was expressed in-house in glycoengineered P. pastoris under the control of the inducible AOX1 promoter.
  • the reporter proteins, single chain insulin fused to the CTP peptide (SCI-CTP) and single chain insulin fused to the highly O-glycosylated peptide of the human TNFR2 ectodomain (SCI- OPEP) were expressed in a GFI1.0 glycoengineered P. pastoris strain (Li et al. 2006), which produced extended a- 1 ,2-mannose containing O-linked chains.
  • the reporter protein human Fc double mutein sequence 1 was expressed in a GFI2.0 glycoengineered P. pastoris strain (Li et al. 2006), which produced O-linked glycans composed primarily of single O-linked mannose residues.
  • GFI1.0 and GFI2.0 glycoengineered strains secreted recombinant protein devoid of ⁇ -mannose and phosphomannose additions.
  • Commercial Leukine ® was purchased from a Pharmacy. This protein was produced in a Saccharomyces cerevisiae yeast strain.
  • Both of the SCI fusion proteins were chosen due to the non-insulin fusion partners being heavily O-glycosylated.
  • a GFI1.0 glycoengineeered background was chosen for these reporter proteins to allow screening for both reduction of extended a-l ,2-mannose polymers and the cleavage of the Man-O-Ser/Thr glycosidic bond.
  • the hFcDM reporter protein was chosen as an example of a different protein class.
  • a GFI2.0 glycoengineered background was chosen to show specific cleavage of the Man-O-Ser/Thr glycosidic bond.
  • Commercial Leukine ® (a S. cerevisiae produced GM-CSF) was chosen as it exemplifies how the LMann can faciliate O-mannose reduction on a non-Pichia pastoris produce glycotherapeutic.
  • Synthetic single chain insulin fusion proteins were generated by fusing either the O- glycosylated carboxyl-terminal peptide (amino acids 136 to 163 of the precursor) of the human chorionic gonadotropin beta-subunit or an O-glycosylated ectodomain fragment (amino acids 221 to 240 of the precursor) of human tumor necrosis factor receptor 2 peptide, and were designated SCI-CTP and CTP-OPEP respectively. Both SCI-CTP and SCI-OPEP were expressed in a GFI 1 .0 glycoengineered P. pastoris strain (Li et al. 2006), which produced extended a-1 ,2- mannose containing O-linked chains.
  • hFcDM sequence 1 Human Fc double mutein (hFcDM) sequence 1 was expressed in a GFI2.0 glycoengineered P. pastoris strain (Li et al. 2006), which produced O- linked glycans composed of a single O-linked mannose residue. Both the GFI1.0 and GFI2.0 glycoengineered strains secreted recombinant protein devoid of ⁇ -mannose and phosphomannose additions.
  • each vector was based upon the previously described vector pGLY2088 (Nett et al. 2012). This latter vector is a roll-in integration vector that targets integration of the vector to the TRP2 locus and contains the bleomycin (ble) resistance gene to confer selection on ZeocinTM.
  • the vector pGLY2088 also possesses an AOX1 inducible expression cassette, which contains functional fragments of P.
  • Partial synthetic open reading frames encoding fragments of the twelve LMann ORFs were generated by Life Technologies/ GeneArt ® (Regensburg, Germany) using the
  • GeneOptimizer ® software and codon-optimized for P. pastoris expression Briefly, fragments possessing from 5' to 3 ' an EcoRl restriction site, DNA encoding codon-optimized amino acid fragments of each LMann as highlighted in Table 1 and SEQ ID NO. l to 12 , DNA encoding an amino acid "GGGGS" linker, DNA encoding a six histidine tag (6xHIS), a stop codon and a Swal restriction site were generated for each LMann.
  • Figure 2 A shows a representative final vector, pGLY12387
  • Figure 2B summarizes the domain structures of the synthetic ORF generated for each of the recombinant LMann.
  • the upper and lower panels, containing amino acid sequences, represent the amino acids added to the N- and C- termini of the amino acid fragments of each of the LMann fragments highlighted in Table 1.
  • the LMann expression vectors pGLY12376 to pGLY12387 were digested with the restriction enzyme Spel to linearize, transformed into the wild-type P. pastoris strain NRRL-Y1 1430 (ATCC, Manassas, VA) by electroporation (Choi et al. 2003) and plated on YSD plates containing 100 ⁇ g/ml ZeocinTM (Life Technologies, Grand Island, NY).
  • Several representative clones potentially expressing each LMann were either screened directly for their ability to hydrolyze O-linked mannose using SCI- CTP. Small scale expression of LMann in P. pastoris
  • Representative clones of each LMann homologue transformed into NRRL-Y1 1430 were grown up in shake flasks containing 50ml BSGY (40 g/L glycerol, 20 g/L soytone, 10 g/L yeast extract, 1 1.9 g/L KH2P04, 2.3 g/L K2HP04, 18.2 g/L sorbitol, 13.4 g/L YNB with ammonium sulfate without amino acids, 8 mg/L biotin) for 72h at 26°C.
  • BSGY 40 g/L glycerol, 20 g/L soytone, 10 g/L yeast extract, 1 1.9 g/L KH2P04, 2.3 g/L K2HP04, 18.2 g/L sorbitol, 13.4 g/L YNB with ammonium sulfate without amino acids, 8 mg/L biotin
  • BSMY the composition of which was identical to BSGY, except that it contained 1 % methanol (containing 0.88 mg/L of PMTi4) in place of the glycerol) prior to resuspension in 5ml BSMY and incubation at 26°C. After 24h an additional charge of methanol to a final concentration of 1% (v/v) was added. The culture supernatants were harvested after a further 24h incubation at 26°C. A control sample was prepared where untransformed NRRL-Y1 1430 was grown and mock-induced in parallel, to provide supernatant devoid of expressed LMann.
  • ⁇ ⁇ of harvested culture supernatant was added to a vial containing ⁇ of 5x reaction buffer (lOOmM sodium acetate, 2mM Zn2+, pH 5.0), 1.5 ⁇ 1 SCI-CTP (3.3mg/ml), 0.5 ⁇ 1 ⁇ ⁇ protease inhibitor cocktail (330mg/L Pepstatin A and 220mg/L Chymostatin in methanol) and the volume brought up to 50 ⁇ 1 with distilled water. Reactions were incubated for 16-24h at 37°C prior to submitting samples for Q-TOF analysis. To reduce the extent of a-l ,2-mannose extensions on the O-linked glycans of SCI-CTP reactions were performed in the presence of both LMann supernatants and recombinant
  • TrMannl For these ⁇ of harvested culture supernatant was added to a vial containing ⁇ ⁇ of purified TrMannl (0.35 mg/ml), 5 ⁇ 1 of lOx reaction buffer (0.2M ammonium acetate, pH6.0 containing lOmM calcium chloride), 1.5 ⁇ 1 SCI-CTP (3.3 mg/ml), 0.5 ⁇ 1 ⁇ protease inhibitor cocktail (330 mg/L Pepstatin A and 220mg/L Chymostatin in methanol) and the volume brought up to 50 ⁇ 1 with distilled water. A control lacking TrMannl was run in parallel, with the volume of distilled water being adjusted accordingly. Reactions were incubated for 16-24h at 37oC prior to submitting samples for Q-TOF analysis.
  • lOx reaction buffer 0.2M ammonium acetate, pH6.0 containing lOmM calcium chloride
  • SCI-CTP 3.3 mg/ml
  • 0.5 ⁇ 1 ⁇ protease inhibitor cocktail 330 mg
  • strains were performed in bioreactors using inoculum seed flasks as described below.
  • the inoculum seed flasks were inoculated from yeast patches (isolated from a single colony) on agar plates into 0.1 L of 4% BSGY in a 0.5-L baffled flask. Seed flasks were grown at 180 rpm and 24°C (Innova 44, New Brunswick Scientific) for 48 hours. Cultivations were done in 1 L (fedbatch-pro, DASGIP BioTools) bioreactors. Vessels were charged with 0.54 L of 0.22 ⁇ filtered 4% BSGY media (with 4 drops/L Sigma 204 antifoam and 50 g/L Maltitol) and autoclaved at 121°C for 60 min.
  • PTM2 salts were injected manually. Completion of the glycerol fed-batch was followed by a 0.5 hour starvation period and initiation of the induction phase. A continuous feed of a 50% v/v methanol solution containing 2.5 mg/L biotin and 6.25 mL/L PTM2 salts was started at a flat rate of 2.16 mL/hr. Injections of 0.5 mL of protease inhibitor solution containing 3.6 mg/mL
  • Pepstatin A and 2.2 mg/mL Chymostatin (in methanol) were added at the start of induction and after each 24 hours of induction time. Additionally, injections of 0.25 mL of 1.9 mg/ml PMTi4 (in methanol) were added each 24 hours of induction. Individual fermentations were harvested after 60 hours of induction. The culture broth was clarified by centrifugation (Sorvall Evolution RC, Thermo Scientific) at 8500 rpm for 40 min and the resulting supernatant was submitted for purification. Purification of active LMaiin
  • Immobilized metal ion affinity chromatography was used for the purification of 6xHis-tagged recombinant LMann enzyme produced in Pichia pastoris.
  • the cell free supernatant sample was transferred to a 10 ml STREAMLINETM Chelating column (GE healthcare, Cat: 17-1280-01) preloaded with nickel ions, at 2.0 ml/min and equilibrated with
  • Intact glycoprotein (SCI-CTP, hFcDM SEQ1 or SCI-OPEP) 50 ⁇ g were dissolved in reaction buffer containing 20 mM sodium acetate, 0.4 mM Zn2+ pH 5.0 and incubated with 3 ⁇ g of LMann enzyme for 16h at 37°C prior to analysis.
  • ReMannI a-l,2-mannosidase
  • 50 ⁇ g of glycoprotein was dissolved in 20mM ammonium acetate pH6.0 containing ImM CaC12 and incubated with 2 ⁇ g of enzyme for 16h at 37°C prior to analysis.
  • Commercial Leukine® (sargramostim) yeast expressed recombinant GM-CSF was subjected to similar enzymatic digest as mentioned above.
  • Mass spectrometric analysis was done in positive ion mode on Accurate-Mass Q-TOF LC/MS 6520 (Agilent technologies, Santa Clara, CA). The protocol used was as previously described (Choi et al. 2012), except that only intact glycoprotein analysis was performed.
  • the O-glycans were released from the reporter protein by ⁇ - elimination under alkaline conditions, processed and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as described previously (Stadheim et al. 2008). SDS-PAGE gels and immunodetection
  • the LMann sequences were cloned into P. pastoris expression vectors which would facilitate expression and secretion recombinant versions of each from this host organism.
  • the yeast expression vector used integrated at the TRP2 loci and used the bleomycin resistance marker to confer resistance to ZeocinTM ( Figure 2A).
  • the lysosomal mannosidase was under the transcriptional control of the methanol inducible AOX1 promoter.
  • the ORF encoding the LMann sequence possessed and N-terminal alpha-mating factor prepro secretion signal, a double FLAG tag, the LMann seqeunce and a C-terminal HIS tag ( Figure 2B).
  • the alpha-mating factor prepro sequence is cleaved to produce a secreted N- terminally FLAG tagged and C-terminally HIS tagged protein.
  • Each of these tags can be used for purification or detection purposes.
  • the LMann encoded amino acids of each vector is highlighted in Table 1 and SEQ ID NOS: 1 through 12. The N- and C-terminal amino acid sequence added to each LMann is depicted in Figure 2B.
  • Table 1 List of LMann homologues used in the current study
  • the O-mannose structures on the recombinant proteins expressed in glycoengineered P. pastoris consist primarily of a-linked mannose. Therefore, a panel of lysosomal mannosidase enzymes were recombinantly expressed and screened for their ability to reduce extended O-linked mannose polymers, in addition to specifically cleaving the Man-O-Ser/Thr glycosidic bond.
  • Table 2 summarizes the preliminary data obtained by screening the supernatants harvested from cultures of P. pastoris which had been transformed with each of the LMann expression vectors. Those homologues reported as having no detectable O-mannosidase activity gave Q-TOF profiles similar to Figure 3B, which in turn is similar to the Q-TOF profile of the SCI-CTP substrate used ( Figure 3A). The GmLMann supernatant gave a Q-TOF profile similar to that for the MtLMann, represented in Figure 3C.
  • Example 3 Enhancement of VvLMann ability to specifically cleave the Man-O-Ser/Thr glycosidic bond.
  • the SCI-CTP was produced in a GFI1.0 glycoengineered background, which produces O-linked glycans with extended a-l,2-mannose chains.
  • the VvLMann was cleaving the Man-O-Ser/Thr, the extent of extended a-1 ,2-mannose chains were reduced using TrMannl.
  • Figures 4C and 4D which were run in parallel but with the latter also including TrMannl, the relative proportion of unglycosylated SCI- CTP increased, thus showing that the VvLMann could more efficiently cleave the Man-O- Ser/Thr glycosidic bond when extended a-l ,2-mannose chains were reduced.
  • the peak area of defined SCI-CTP molecular forms were assessed using the Agilent Chameleon software to calculate peak area.
  • the data provided in Table 3 provides an estimation of the relative degree of O-mannosylation of individual or collated species based on the peak area(s) of the Q-TOF profile (Note: the 9051 peak mass value corresponds to the lowest peak mass in range, additional peak masses can be obtained by adding 162 amu).
  • Vv-, Gm-, Mt-LMann and HsLMann have similar O-glycan reduction activity
  • subsequent examples will expand only on characterizing the VvLMann, with the assumption that the other enzymes behave in a similar fashion.
  • Figure 9 depicts the digestion of O-linked glycans under vaiying co-ion conditions.
  • the Q-TOF profile of the starting SCI-CTP can be seen in Figure 9A, and indicates that the molecule is heavily O-glycosylated, and that the mass for unglycosylated intact SCI-CTP is not detected.
  • Q-TOF analysis of SCI-CTP in the presence or absence of adding additional zinc ions shows that the majority of the O-linked glycans have been removed, as indicated by the prominent species detected being unglycosylated SCI-CTP (black arrow).
  • Table inset in Figure 9 was compiled.
  • the data in Table 4 provides an estimation of the relative degree of O-mannosylation of individual or collated species based on the peak area(s) of the Q-TOF profile (Note: the 9051 peak mass value corresponds to the lowest peak mass in range, additional peak masses can be obtained by adding 162 amu).
  • the peak area percentage values summarized in Table 4 indicates that prior to treatment 100% of the SCI-CTP possessed more than one O-linked mannose residue.
  • the data in Table 5 was compiled using the Agilent Chameleon software to calculate peak area.
  • the data provides an estimation of the relative degree of O-mannosylation of individual molecular species based on the peak areas of the Q-TOF profile.
  • the peak area percentage values summarized in Table 5 indicates that prior to treatment >30% of the hFcDM possessed more than one O-linked mannose residue.
  • the data in Table 6 was compiled (Note the 8517 peak mass value corresponds to the lowest peak mass in range, additional peak masses can be obtained by adding 162 amu).
  • the data provided in Table 6 represents an estimation of the relative degree of O-mannosylation of individual or collated molecular species based on the peak areas of the Q-TOF profile.
  • the peak area percentage values summarized in Table 6 indicates that prior to treatment 100% of the SCI-OPEP possessed more than three O- linked mannose residues.
  • Example 7 Assessment of LMann to remove O-linked mannose from a non-P. pastoris produced O-glycosylated protein.
  • Leukine ® is a commercially available recombinant form of granulocyte macrophage colony-stimulating factor (GM-CSF) produced in the yeast Saccharomyces cerevisiae.
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • the data in Table 7 was compiled using the Agilent Chameleon software to calculate peak area (Note: the 14755 peak mass value corresponds to the lowest peak mass in range, additional peak masses can be obtained by adding 162 amu).
  • the table provides an estimation of the relative degree of O-mannosylation of individual or collated molecular species based on the peak areas of the Q-TOF profile.
  • the peak area percentage values summarized in Table 7 indicates that prior to treatment greater than 52% of the Leukine ® possessed more than one O-linked mannose residue. However, following incubation of Leukine ® with VvLMann greater than 95% of the molecular forms of Leukine ® were unglycosylated, with the remaining forms possessing primarily a single O-linked mannose residue.
  • Example 8 Production of subdomain variants of recombinant LMann.
  • LMann is a large protein, typically a heterodimer around 150kDa, which later self- dimerizes.
  • the heterodimer is a cleavage product of the LMann precursor peptide. It is this latter single peptide precursor that has been used to exemplify the utility of LMann to remove O-linked mannose. It is foreseeable that a processed dinieric form of the enzyme may be more efficient at removing O-linked mannose on particular proteins due to their differing structural
  • Forms V and VI represent recombinants forms of the enzymes were the N- or C-terminal subdomains have been expressed independently, each with a C-terminal HIS-tag to facilitated purification.
  • Forms V and VI may be used independently of combined for O-mannose removal activity. To date Form I has been able to efficiently remove O-linked mannose from a number of O-mannosylated proteins but it is anticipated that particular O-mannosylated proteins may be more efficiently processed with a subset of Forms II to VI.
  • Example 9 Reduction of O-linked mannose by co-secretion of LMann and the O- glycosylated glycoprotein of interest.
  • Co-secretion of LMann in parallel to the secreted O-mannosylated glycoprotein of interest can be used to reduce the extent of O-linked mannose on the glycoprotein, prior to its purification from the culture broth.
  • the LMann expression vectors described previously can be used directly to transform into a strain already expressing the protein of interest.
  • the LMann integrates into the TRP2 loci using Zeocin for selection.
  • the current vectors can integrate into the AOX1 promoter loci, using the restriction enzyme Pmel to linearize the DNA prior to transformation into the yeast strain.
  • the current vectors can be modified to increase their utility by replacing the Bleomycin resistance cassette with another selectable marker to facilitate selection of a host strain that already possesses Zeocin resistance.
  • selectable markers are the URA5 blaster counter-selectable marker or any other auxotrophic marker, wherein the host strain is deficient in the activity of that essential enzyme.
  • another dominant marker can be used which confers resistance to a normally toxic compound. Such markers could confer resistance to hygromycin, kanamycin, G418, nouresothricin, arsenite etc.
  • LMann The introduction of LMann outlined above can result in multiple integrations of the LMann expression cassette, and is referred to as roll-in integration.
  • An alternative approach is to introduce the LMann expression cassette using a knock-in vector strategy were only one copy of the LMann expression cassette is integrated into the yeast genome. Examples of the structures of knock-in vectors are common in the literature and can be seen exemplified in (Nett et al. 2005).
  • An alternative approach to introducing the LMann after the strain has been modified to produce the O-mannosylated protein of interest, is to either introduce the LMann prior to or while introducing the construct for expression of the glycoprotein of interest.
  • the LMann is under the control of the methanol inducible AOX1 promoter, similar to the putative glycoprotein of interest. By changing the promoter on either or both, it is possible to regulate the relative expressional timing of each. Alternatively constitutive expression of each can be desirable, where each is under the expressional control of a constitutive promoter such as the GAPDH promoter.
  • the co-secretion of the LMann along with the O-mannosylated protein of interest results in the production of a glycoprotein of interest with reduced O-mannosylation, which in turn avoids the need to perform an in vitro de-O-mannosylation step post-purification of the protein.
  • O-mannose removal can be manipulated. For instance, varying the pH of the media can be used to enhance or reduce the activity of the LMann.
  • GmLMann (SEQ ID NO: 3)
  • GpLMann (SEQ ID NO: 4) A G Y E T C P M V Q P G M L N V H L V A H T H D D V G W L K T V D Q Y Y W G I H N D L Q Q A G V Q Y I L D S V I S A L L A E P T R R F V Y V E M A F F S R W W H Q Q T N E T Q E V V V R R L V R Q G R L E F A N G G W V M N D E A A T H Y G A I V D Q M T L G L R F L E D T F G S D G R P R V A W H I D P F G H S R E Q A S L F A Q M G F D G V F F G R I D Y Q D K L V R K K R E M E L V W R A S A S L K A P A A D L F T G V L P N N Y G P P E G L C W D V L C A D P P V V D D D
  • HsLmann (SEQ ID NO: 5)
  • MtLMann (SEQ ID NO: 6)
  • MmLMann (SEQ ID NO: 7)
  • RcLMann (SEQ ID NO: 8)
  • SsLMann (SEQ ID NO: 9)
  • TcLMann (SEQ ID NO: 10)
  • VvLMann (SEQ ID NO: 12)
  • Rhodanine-3 -acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des procédés in vitro et in vivo d'utilisation de mannosidases lysosomaux pour éliminer des résidus de mannose liés à O de glycoprotéines intactes, consistant à hydrolyser la liaison Man-α-O-Ser/Thr glycosidique.
PCT/US2014/047120 2013-07-25 2014-07-18 Procédé de réduction de l'étendue d'o-mannosylation de glycoprotéines Ceased WO2015013116A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361858170P 2013-07-25 2013-07-25
US61/858,170 2013-07-25

Publications (1)

Publication Number Publication Date
WO2015013116A1 true WO2015013116A1 (fr) 2015-01-29

Family

ID=52393757

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/047120 Ceased WO2015013116A1 (fr) 2013-07-25 2014-07-18 Procédé de réduction de l'étendue d'o-mannosylation de glycoprotéines

Country Status (1)

Country Link
WO (1) WO2015013116A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
US10680697B2 (en) 2016-05-12 2020-06-09 Huawei Technologies Co., Ltd. Channel state information feedback method, base station, terminal device, and system
US10724013B2 (en) 2013-07-04 2020-07-28 Glykos Finland Oy O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6890748B2 (en) * 2000-07-26 2005-05-10 Large Scale Biology Corporation Production of lysosomal enzymes in plants by transient expression
US20090170159A1 (en) * 2005-11-15 2009-07-02 Glycofi, Inc Production of glycoproteins with reduced o-glycosylation
WO2012042387A2 (fr) * 2010-09-29 2012-04-05 Oxyrane Uk Limited Démannosylation des n-glycanes phosphorylés
US20120225453A1 (en) * 2010-07-30 2012-09-06 Withers Iii Sydnor T Systems and methods for the secretion of recombinant proteins in gram negative bacteria
US8298811B2 (en) * 2003-02-20 2012-10-30 Glycofi, Inc. Expression of Class 2 mannosidase and Class III mannosidase in lower eukaryotic cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6890748B2 (en) * 2000-07-26 2005-05-10 Large Scale Biology Corporation Production of lysosomal enzymes in plants by transient expression
US8298811B2 (en) * 2003-02-20 2012-10-30 Glycofi, Inc. Expression of Class 2 mannosidase and Class III mannosidase in lower eukaryotic cells
US20090170159A1 (en) * 2005-11-15 2009-07-02 Glycofi, Inc Production of glycoproteins with reduced o-glycosylation
US20120225453A1 (en) * 2010-07-30 2012-09-06 Withers Iii Sydnor T Systems and methods for the secretion of recombinant proteins in gram negative bacteria
WO2012042387A2 (fr) * 2010-09-29 2012-04-05 Oxyrane Uk Limited Démannosylation des n-glycanes phosphorylés

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10724013B2 (en) 2013-07-04 2020-07-28 Glykos Finland Oy O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof
US10513724B2 (en) 2014-07-21 2019-12-24 Glykos Finland Oy Production of glycoproteins with mammalian-like N-glycans in filamentous fungi
US10680697B2 (en) 2016-05-12 2020-06-09 Huawei Technologies Co., Ltd. Channel state information feedback method, base station, terminal device, and system

Similar Documents

Publication Publication Date Title
EP1597379B2 (fr) Endomannosidases dans la modification de glycoproteines d'eukaryotes
Parsaie Nasab et al. A combined system for engineering glycosylation efficiency and glycan structure in Saccharomyces cerevisiae
JP4464269B2 (ja) 哺乳類型糖鎖生産メチロトロフ酵母
Kainz et al. N-glycan modification in Aspergillus species
Dragosits et al. Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool
WO2011134643A1 (fr) Glycosylation améliorée de protéines dans des cellules hôtes
KR20160030127A (ko) O-만노실트랜스퍼라제 결핍 사상균 세포 및 이의 이용 방법
WO2015013116A1 (fr) Procédé de réduction de l'étendue d'o-mannosylation de glycoprotéines
Hopkins et al. Elimination of β-mannose glycan structures in Pichia pastoris
Gomathinayagam et al. In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins
JP5967725B2 (ja) 複合型糖鎖加水分解酵素
Hopkins et al. A practical approach for O-linked mannose removal: the use of recombinant lysosomal mannosidase
US20260042998A1 (en) Glyco-Engineered Yeast for Therapeutic Protein Production
AU2013363266A1 (en) pmt2, och1, pmt5 Mutant Cells
CA3022498C (fr) Moyens et procedes pour la production de glycanes complexes issus d'hotes fongiques modifies
US20250101483A1 (en) Glycoengineering of thermothelomyces heterothallica
JP6912781B2 (ja) 操作された真菌宿主に由来する複合グリカンを産生するための手段および方法
Hopkins et al. Elimination of diaminopeptidase activity in Pichia pastoris for therapeutic protein production
Zha et al. Production of monoclonal antibodies in glycoengineered Pichia pastoris
Lerouge et al. Elodie Mathieu-Rivet1†, Martin Scholz2†, Carolina Arias3†, Flavien Dardelle1#, Stefan Schulze2#, François Le Mauff4, Gavin Teo4, Ana Karina Hochmal2, Amaya Blanco-Rivero3, Corinne Loutelier-Bourhis5, Marie-Christine Kiefer-Meyer1, Christian Fufezan2, Carole Burel1
EP2563902A1 (fr) Glycosylation améliorée de protéines dans des cellules hôtes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14829849

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14829849

Country of ref document: EP

Kind code of ref document: A1