Industrial production of Human Milk Oligosaccharides

through industrial biotechnology

Prof. Wim Soetaert

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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

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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

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• 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?

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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

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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

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Metabolically engineered microbes

Fermentation

Down-stream processing

Microbesat work

Specialty carbohydrate

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Microbial synthesis of building blocks

Sucrose

Carbohydrate building blocks

Microbesat work

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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

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Transfer into the base strain

Sucrose

Specialty carbohydrate

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Biochemical pathway assembly

Selection of best strain from the library

carbohydrate

carbohydrate

carbohydrate

carbohydrate

carbohydrate

carbohydrate

carbohydrate

carbohydrate

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Strain development

Fermentation technology

Synthetic Biology &

Automation

AnalyticsBio-

informatics & Statistics

Enzymeengineering

Confidential

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Technology platform for high throughput development ofspecialty carbohydrate production strains

bugMetabolic engineering of the production organism

Process

Product

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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

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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

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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

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HMO strain & process improvement

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Final titer(g/L)

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Scale-up of the fermentation process

Product

• Development of down-stream processing of fermentation broth

• Product recovery and purification

• Product conditioning

Integrated technology development

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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

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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

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Integrated technology development

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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

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Pubmed - human milk oligosaccharide publication count per yr

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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

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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

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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