Northwest Glycosciences Workshop

1
Yarrowia lipolytica a versatile expression
platform for the production of therapeutic
glycoproteins
Adriana Botes Oxyrane UK Ltd Oxyrane Belgium
NV VIB (Ghent) INRA (Grignon)
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Contents

• Introduction
• Distinguishing characteristics of Y. lipolytica

• Development of Y. lipolytica as an expression
  platform for multiple applications

• Glycoengineered Y. lipolytica strains for the
  production of therapeutic glycoproteins
• Targeted Enzyme Replacement Therapies for
  Lysosomal Storage Diseases
• Next generation Monoclonal antibodies with
  modulated effector functions

• Future developments in glycoprotein therapeutics
• Strategies to design produce optimised
  glycofroms of therapeutic glycoproteins
• The role benefits of a regional glycosciences
  network

3
Distinguishing characteristics of Y. lipolytica

• Suitablility as industrial production host for
  products intended for human use
• GRAS status strictly aerobic, non-pathogenic
• industrial production of citric acid, aroma
  compounds DCAs
• FTO entire genome sequenced (Genome evolution
  in yeasts 2004 Nature 430, 34-44)

• Y. lipolytica diverges greatly from other
  ascomycetous yeasts
• Dipodascacea family - only known species in its
  genus (Bigey, Tuery et al. 2003)
• FTO in most application areas covered for
  conventional methylotrophic yeasts

• Adaptations to utilise hydrophobic substrates
• Surfactant- mediated transport- biocatalysis
  applications
• Direct interfacial transport- protrusions on cell
  surface YSD applications
• Expanded membrane capacity-Expression of integral
  membrane proteins (GPCRs)

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Adaptations to utilise hydrophobic substrates

• Surfactant-mediated transport
• Direct interfacial transport
• Cell surface is modified by the production of
  protrusions (PT) on top of which HS/ lipid
  droplets (LD) bind
• ? apolar properties of cell surface,
• ? HS droplet sizes ? HS droplet numbers
• Improves contact between HS and cells

A Glucose medium B min. oleic acid medium, 2
hrs C min. oleic acid medium, 18 hrs D E the
number of PTs LDs ? as an adaptative response
to hydrophobic substrates (Mlícková et al. 2004)
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Distinguishing characteristics of Y. lipolytica

• Suitablility as industrial production host for
  products intended for human use
• GRAS status strictly aerobic, non-pathogenic
• industrial production of citric acid, aroma
  compounds DCAs
• FTO entire genome sequenced (Genome evolution
  in yeasts 2004 Nature 430, 34-44)

• Y. lipolytica diverges greatly from other
  ascomycetous yeasts
• Dipodascacea family - only known species in its
  genus (Bigey, Tuery et al. 2003)
• FTO in most application areas covered for
  conventional methylotrophic yeasts

• Adaptations to utilise hydrophobic substrates
• Surfactant- mediated transport- biocatalysis
  applications
• Direct interfacial transport- protrusions on cell
  surface YSD applications
• Expanded membrane capacity-Expression of integral
  membrane proteins (GPCRs)

6
Distinguishing characteristics of Y. lipolytica

• Distinguished for capacity to secrete proteins
  into the medium
• Proteases, Lipases, Phosphatases, RNase,
  Esterase
• 1-2 g/L under inducing conditions

• Overlapping characteristics with basidiomycetes
  and higher eukaryotes
• Greater complexity of the secretory pathway as is
  found in fungi mammals
• Translocation apparatus is largely devoted to
  co-translational translocation of nascent
  peptides through the ER as present in mammalian
  cells

Excellent host for the production of complex
heterelogous proteins
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Production of a homologous glycoprotein Lip2p

• Dispersed, multi-copy integration, inducible
  Pox2 promoter

MW 25h 41h 50h 65h 74h 89h 97h 163h MW kDa
107
76
52
37
27
19
SDS-PAGE 5 µl supernatant
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Production of a heterologous glycoprotein

• 4 copies, targeted integration, Pox2 promoter

Process conditions Cultivation time Sample
Un-optimised fed-batch system
Oxyrane semi-optimised fermentation
12h
44h
20h
14h
12h
44h
20h
14h
1 2 3 4 5 6 7 M 8 9 10 11 12 13 14
220 kDa
70 kDa
60 kDa
50 kDa
40 kDa
30 kDa
gt2 g/L lt 30 hours
25 kDa
20 kDa
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Contents

• Introduction
• Distinguishing characteristics of Y. lipolytica

• Development of Y. lipolytica as an expression
  platform for multiple applications

• Glycoengineered Y. lipolytica strains for the
  production of therapeutic glycoproteins
• Targeted Enzyme Replacement Therapies for
  Lysosomal Storage Diseases
• Next generation Monoclonal antibodies with
  modulated effector functions

• Future developments in glycoprotein therapeutics
• Strategies to design produce optimised
  glycofroms of therapeutic glycoproteins
• The role benefits of a regional glycbiology
  network

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Development of YL as expression platform for
multiple applications
Unique uptake mechanisms of HS apolar cell
surface Expression of hydrophobic membrane
proteins
Whole cell biocatalysis
Antibody engineering (Yeast Surface Display)
Proteins that form protrusions on cell surface
Surface display, episomal vectors, yeast mating
Expression of Secreted heterologous enzymes
proteins
Efficient secretion of complex heterologous
proteins
Sequential targeted integration to optimise copy
number
Tightly regulated promoters (Tet-ON/ Tet-Off)
Minimal glycosylation (Man3), ?UPR (Hac1,
chaperones) Expanded membrane capacity
Expression of hydrophobic membrane
proteins Unique lipid composition of membranes
Expression of Integral membrane proteins as drug
targets (GPCRs)
Glycoengineering Man8, Man5 Man3 Man-6P,
non-fucosylated Regulation of expression levels
of multiple genes ?UPR (Hac1, chaperones) In
vitro glycan elaboration
Expression of Secreted glycoproteins with
homogeneous/optimal glycan structures
Co-translational modification, mammalian-like
secretory pathway
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Contents

• Introduction
• Distinguishing characteristics of Y. lipolytica

• Development of Y. lipolytica as an expression
  platform for multiple applications

• Glycoengineered Y. lipolytica strains for the
  production of therapeutic glycoproteins
• Targeted Enzyme Replacement Therapies for
  Lysosomal Storage Diseases
• Next generation Monoclonal antibodies with
  modulated effector functions

• Future developments in glycoprotein therapeutics
• Strategies to design produce optimised
  glycofroms of therapeutic glycoproteins
• The role benefits of a regional glycosciences
  network

12
Enzyme Replacement Therapies for LSDs

• Glycans targeting the Macrophage Mannose Receptor
• ß-Glucocerebrosidase (Gauchers disease)

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Enzyme Replacement Therapies for LSDs

• Glycans targeting the Mannose 6-Phosphate Receptor

• a-Galalactosidase (Fabrys disease)
• a-Glucosidase (Pompes disease)
• a-L Iduronidase (MPS 1)
• Sulfatases (MPS II, IIIA, IIID, VI)

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Monoclonal antibodies (IgGs)
Non-fucosylated mAbs display enhanced ADCC
response
Absence of core fucose results in 50 100 x ?
in binding to Fc?RIIIa receptor
The core heptasaccharide present in normal
human IgG
Diantennary oligosaccharide structure attached to
IgG-Fc at Asn297.
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Monoclonal antibodies (IgGs)
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Contents

• Introduction
• Distinguishing characteristics of Y. lipolytica

• Development of Y. lipolytica as an expression
  platform for multiple applications

• Glycoengineered Y. lipolytica strains for the
  production of therapeutic glycoproteins
• Targeted Enzyme Replacement Therapies for
  Lysosomal Storage Diseases
• Next generation Monoclonal antibodies with
  modulated effector functions

• Future developments in glycoprotein therapeutics
• Strategies to design produce optimised
  glycofroms of therapeutic glycoproteins
• The role benefits of a regional glycosciences
  network

17
Future Developments in Glycoprotein therapeutics

• Strategies to design produce homogeneous,
  optimised glycofroms of therapeutic glycoproteins
• In vivo glyco-engineering of production hosts is
  time-consuming and affects the growth phonotype-
  limited glycan library size
• Combined in vivo / in vitro approaches allows
  generation of larger glycan libraries for
  screening of glycoforms with optimal therapeutic
  performance

• Glyco-engineered hosts to produce basic glycan
  scaffolds
• Further in vitro elaboration of the glycan
  scaffolds with glycosyltransferases
• Requires off the shelf libraries of recombinant
  glycosyltransferases, know-how databases of
  available enzymes activities/specificities

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Future Developments in Glycoprotein therapeutics

• Chemo-enzymatic synthesis of homogeneous
  N-glycoproteins
• Transglycosylation by endo-ß-N-acetylglucosaminida
  se (ENGases)
• Advantage en block transfer of oligosaccharides
  in a single step
• Disadvantage low yields with natural
  oligosaccharides BUT effective with activated
  synthetic sugar oxazolines
• Requires a readily available library of ENGases
• Enzyme engineering to obtain glycosynthase
  activity
• Scalable affordable chemical synthesis of sugar
  oxazolines

H. Ochiai et al., 2008. J. Am Chem. Soc., 130
(41) 13790-13803
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Future Developments in Glycoprotein therapeutics

• Chemical glycosylation of cysteine surface
  residues in proteins
• introduces glycans at unique cysteine surface
  residues in proteins and thus produces
  semi-synthetic neo-glycoproteins
• Requires mutation of specific residues (e.g.
  Asn) in recombinant proteins to cysteine
• Requires off the shelf micro-arrays of glycans
  

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The role benefits of a regional glycobiology
network

• Service facility for the analysis of N- and O-
  glycans (infrastructure)
• Consultancy service / forum for discussions with
  experts to advise on most appropriate strategies
  for chemo-enzymatic or chemical synthesis of
  N-glycoproteins
• Network / Database of regional national
  research groups, companies service
  organisations involved in glycosciences
• Access to readily available glycosciences tools
  (and know-how transfer)
• Glycosyltransferases / ENGases
• Synthetic carbohydrate libraries (including
  sugar oxazolines)
• Research funding for projects that will serve the
  glycosciences community
• Project proposals from researchers companies
  approved by a glycosciences network committee-
  results and tools developed available under
  non-exclusive license to companies
• Highly trained students that can exit academia
  into companies with a glycosciences focus