Industrial production of Human Milk Oligosaccharides...
41
Industrial production of Human Milk Oligosaccharides through industrial biotechnology Prof. Wim Soetaert
Transcript of Industrial production of Human Milk Oligosaccharides...
Industrial production of Human Milk Oligosaccharides
through industrial biotechnology
Prof. Wim Soetaert
2
Inbiose at a glance
Confidential
Core competence in the development and production of specialty carbohydrates through
industrial biotechnology
Proprietary production platformbased on fermentative synthesis
of specialty carbohydrates (cell factory)
Access to pilot and production facilities
Capacity up to 100tpa
Experienced team with industrial and academic background
“Specialty carbohydrates”Started in 2013
Ghent University spin-offFast growing: >30 FTE in 2016
3
Carbohydrates are everywhere
From the simplest form
typically used as food
To very complex structures
used as an active ingredient
glucose
Specialty carbohydrate
Carbohydrates are everywhere
4
• Very complex carbohydrates with unconventional structures
• Rare in nature or difficult to impossible to produce
• Price range: 10 €/kg – 1.000 €/kg (reference sugar: 0.5 €/kg)
• Quantities: 1 kg – 10.000 tpa
• High-end applications in pharmaceuticals, nutraceuticals,
cosmetics, chemicals, plant protection,...
What are specialty carbohydrates?
5
6
What are specialty carbohydrates?
Carbohydrates consisting of unconventional building blocks:
Example: Human Milk Oligosaccharides
L-Fucose Sialic acid Glucose GalactoseGlucosamine
Disialyl-Lacto-N-Tetraose (dSLNT) as an exampleof a very complex specialty carbohydrate
7
O
OHOOH
OH
OH
OOHO
OH
OH
O
OH
O
NH
O
CH3
O
OOH
O
OH
OH
O
NH
OH
OH OH
O
CH3
O
OH
OH
O
NH
OH
OH OH
O
CH3
O
OH
OH
LactoseLacto-N-Tetraose
N-acetylneuraminate
Occ
urr
ence
sucrose
lactose
chitin
Complexity/difficulty to produce
N-acetylglucosamine
glucose
fructose
galactose
L-fucosesialic acid
mannose
L-ribose
xylose
D-ribose
deoxyribose
LactoNbioselactoNtetraose
Lewis X
FOS
What are specialty carbohydrates?
Pri
ce
Complexity/difficulty to produce
sucrose lactose
chitin
HMO
chitosan
N-acetylglucosamine
glucose fructose
galactose
L-fucose
sialic acid
mannose
L-ribose
XOSxylose
D-ribose
Deoxyribose
GOS FOS
lactoNbiose
lactoNtetraose
Lewis X
What are specialty carbohydrates?
Extraction
Microbial synthesis
Chemical synthesis
Enzymaticsynthesis
Specialty carbohydrate production
Natural source required, availability problemsHigh price volatility due to natural variabilityComplex extraction procedures
Very complex synthesis and costly substratesLow yieldsUse of toxic chemicals and high waste generation
Simple and cheap Only for simple one-step conversionsEquilibrium can be unfavourable
Very complex carbohydrates produced in one stepCheap and readily available substratesEnvironmentally sustainable
Confidential
Inbiose production platform
11
Metabolically engineered microbes
Fermentation
Down-stream processing
Microbesat work
Specialty carbohydrate
12
Microbial synthesis of building blocks
Sucrose
Carbohydrate building blocks
Microbesat work
13
Microbial assembly of the building blocks
Assembly of the different monosaccharide building blocks
into an oligosaccharide
Pathway engineering
Biochemical pathway assembly
Cloned genes with tunable elements such as promoters and ribosome binding sites
Assemble in an artificial chromosome
14
Transfer into the base strain
Sucrose
Specialty carbohydrate
15
Biochemical pathway assembly
Selection of best strain from the library
carbohydrate
carbohydrate
carbohydrate
carbohydrate
carbohydrate
carbohydrate
carbohydrate
carbohydrate
16
Strain development
Fermentation technology
Synthetic Biology &
Automation
AnalyticsBio-
informatics & Statistics
Enzymeengineering
Confidential
17
Technology platform for high throughput development ofspecialty carbohydrate production strains
bugMetabolic engineering of the production organism
Process
Product
18
Development of the fermentation process
Development of the product recovery process
Integrated technology development
Bug
• Biochemical pathway design
• Expression of suitable genes in the production host
• Strain selection for optimal producer organism
19
Identify the optimal biosynthesis
pathway in the production hostScreen biosynthesis associated genes and
proteins in DNA and protein databases
ATGGTTGCAGTAGCGATAGC
GCCTTTATAAAT
GCTATAGATCT
GATCTTCGGAA
CTGAGCTTCAA
CTATGCGGCTTA
TAATATTTATTCCAAA CC
TTATCCTTCTCGGAGGCTCT
AGGGGCCTTAAAATTCCCGA
GCSSDSGLYKCRYGYRSIGFG
SSESRDLSFNSEIYAAYRRLI
FIPNSLSFSEALGALKFPNSM
LKKIDPAQTESWKRLGRHYDG
VKDLAMDDLFQQDPDRFKRLS
MAFKDMVVDVSKNRITDKTLA
LLMDLARETGVKEAIAMMFRG
DRINETEGRSVLHVALRNLAD
TPVLVDGKDVMPGVRGVLEDR
Pathway library assembly
Integrated technology development
Process
• Development of the fermentation process
• Optimisation of yield and production rate
• Scale-up of the fermentation process
20
Time
Tite
r
Integrated technology development
23 mg/L 182 mg/L2,3 g/L
5,7 g/L
16 g/L
54 g/L
0
10
20
30
40
50
60
HMO strain & process improvement
21
Final titer(g/L)
22
Scale-up of the fermentation process
Product
• Development of down-stream processing of fermentation broth
• Product recovery and purification
• Product conditioning
Integrated technology development
23
24
Scale-up of the down-stream process
Confidential
Build-up of production capacity
Two new 15 m3 fermentersare operational
HMO Production capacity: 100 tonnes per year
25
Industrial biotechnology process hall
Industrial biotechnology process hall
Industrial biotechnology process hall
Industrial biotechnology process hall
Actiefoto’s
Our technology in a nutshell
Sucrose or glucose
Cell factory HMOs
33
Integrated technology development
34
35
Glucose
• Human mother’s milk composition:
– Protein: 8 g/l
– Fat: 42 g/l
– Lactose: 70 g/l
– Human Milk Oligosaccharide: 15 g/l
• Inbiose’s first target is 2’fucosyllactose as the most abundant HMO
Galactose
Fucose
Human Milk Oligosaccharides
Human Milk Oligosaccharides
Prebiotics Functional foods Feed/Pet careInfant milk
formula
1st market
Confidential
…
HMOs are currently our main targets
Confidential37
41 39
78
100
0
20
40
60
80
100
120
19
53
19
57
19
62
19
65
19
68
19
71
19
74
19
77
19
80
19
83
19
86
19
89
19
92
19
95
19
98
20
01
20
04
20
07
20
10
20
13
20
16
Pubmed - human milk oligosaccharide publication count per yr
1 1 1 1 1 1 0 0 1 1 1 1
4 5
15
21
28
3432
0
5
10
15
20
25
30
35
40
19
98
19
99
20
00
20
01
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
20
11
20
12
20
13
20
14
20
15
20
16
HMO Applications – Patent Families by Year
Confidential38
Enabling “Closest to breast milk”
Enabling Health Benefits to the infant
2’FL is our first HMO
Confidential
39
Strategic and technology fitScience• 50 yrs in (early life) nutrition (proteins,
cultures, pre- & probiotics, hydrocolloids, enzymes)
• Leader in microbiome scienceProduction• Strong position in large scale fermentation,
carbohydrate production and DSPMarket reach• Global market access and reach with local
support• Can supply customers with scale, supply
security and good safety track record
Inbiose - DuPont Partnership
Licence and JDA• DuPont to commercialize 2’FL and
possibly 2 other fucosylated HMO’s for Human Nutrition
• DuPont to lead up-scaling, manufacturing and sales
• Inbiose is supporting customers with samples until commercial launch by DuPont
• Parties working diligently to support customers to launch new products with HMO’s
40
In a nutshell
• Generic and scaleable technology platform for producing HMOs
through industrial biotechnology
• Inbiose can produce any complex carbohydrate for which a
biotechnological pathway can be designed
• Inbiose can quickly develop new processes through its base strains
• Inbiose process starts from cheap substrates and delivers
high yields and productivities
• Inbiose can currently produce 100 tpa of HMOs
2016© Inbiose NV
www.inbiose.com
Turning specialty carbohydrates
into an industrial reality
