Nanobodies® -

Inspired by nature

Double digit-titers and high product quality of Nanobodies®

Manu De Groeve, PhD

Scientist CMC-USP Process Development

Pichia 2014 conference

March 2 – 5, 2014 | San Diego CA, USA

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Forward looking statements

Certain statements, beliefs and opinions in this presentation are forward-looking, which reflect the Company or, as appropriate, the Company directors’ current expectations and projections about future events. By their nature, forward-looking statements involve a number of risks, uncertainties and assumptions that could cause actual results or events to differ materially from those expressed or implied by the forward-looking statements. These risks, uncertainties and assumptions could adversely affect the outcome and financial effects of the plans and events described herein. A multitude of factors including, but not limited to, changes in demand, competition and technology, can cause actual events, performance or results to differ significantly from any anticipated development. Forward looking statements contained in this presentation regarding past trends or activities should not be taken as a representation that such trends or activities will continue in the future. As a result, the Company expressly disclaims any obligation or undertaking to release any update or revisions to any forward-looking statements in this presentation as a result of any change in expectations or any change in events, conditions, assumptions or circumstances on which these forward-looking statements are based. Neither the Company nor its advisers or representatives nor any of its parent or subsidiary undertakings or any such person’s officers or employees guarantees that the assumptions underlying such forward-looking statements are free from errors nor does either accept any responsibility for the future accuracy of the forward-looking statements contained in this presentation or the actual occurrence of the forecasted developments. You should not place undue reliance on forward-looking statements, which speak only as of the date of this presentation.

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Outline

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Ablynx and the Nanobody platform

Nanobody manufacturing in Pichia

Overview

Host creation

USP

YIELD QUALITY

Design of Experiments (DoE) based Process Development

Case studies

DSP

Analytics

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Company highlights

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• ~ €200M in cash estimated at 31st December 2013

• ~ €20-25M net cash burn estimated for the full year 2013

Products

Partners

• Pioneer in next generation biologics – Nanobodies®

• >500 granted and pending patents •

• ~30 programmes – seven in clinical development

• Two clinical proof-of-concepts

• >800 healthy volunteers and patients treated with Nanobodies

•

• AbbVie, Boehringer Ingelheim, Eddingpharm, Merck & Co,

• Merck Serono and Novartis

• >€300M in non-dilutive cash received to date

Financials

• Drug discovery and development company - Ghent, Belgium

• NYSE Euronext Brussels (ABLX)

• 49M shares outstanding (52M fully diluted)

• 280 employees

Technology

Corporate

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Nanobodies – proven single variable domain approach

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Camelidae family has both forms

Conventional antibody

• Heavy and light chains

• Both chains required for antigen

binding and stability

• Large size and relatively low

formatting flexibility

• Administered through injection

Heavy-chain antibody

• Only heavy chains

• Full antigen binding capacity

and very stable

VH

VL CL

CH1

CH3

CH2

VHH

Ablynx’s Nanobody®

• Small (1/10 size of a mAb)

• Flexible formatting

• Highly potent, robust and stable

• Broad target applicability

• Multiple administration routes

• Ease of manufacture

• Speed of discovery

VHH

CH3

CH2

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Nanobodies – uniqueness and competitive advantages

Flexible formatting: multivalent, multi-specific, bi-paratopic

Nanobodies

Broad target applicability, including challenging targets such

as GPCRs and ion channels

Robustness allows for alternative delivery such as nebulisation

Half-life engineering technology to achieve desired properties

(acute vs chronic diseases) (T1/2 from 2h to 20 days)

Excellent manufacturing (yeast and bacteria), high concentration

formulations and low viscosity (excellent syringeability)

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Nanobody discovery process

Conventional

antibodies

Select Nanobodies

of interest

Immunize llama

with antigen Draw blood 6–12

weeks later

Clinical trials

VHH

Ablynx’s

Nanobody®

VHH

CH3

CH2

Manufacture in

micro-organisms

plus half-life

extension

(HLE)

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Format Nanobody to achieve

desired properties

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Anti-RSV – ALX-0171 – 1st inhaled trivalent Nanobody

• Phase I safety study in healthy volunteers successfully completed

• additional pre-clinical and Phase I studies on-going

• first-in-infant study expected to start in H2 2014

• potential transformational treatment for RSV infection in infants

Anti-RANKL – ALX-0141 – bivalent Nanobody with T1/2 extension

• Phase I study successfully completed

• exclusively licensed to Eddingpharm in Greater China

Anti-IL-17A/F – ALX-0761 – bi-specific Nanobody with T1/2 extension

• pre-clinical POC achieved and Phase I study on-going

• Merck Serono has an exclusive license to the programme

Anti-IL-6R – ALX-0061 – monovalent Nanobody with T1/2 extension

• Phase II POC achieved in patients with RA

• global exclusive licensing deal with AbbVie

• Ablynx responsible for next phases of development

• opportunity for differentiation in RA and SLE

Programmes in Phase I and II clinical development

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Outline

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Ablynx and the Nanobody platform

Nanobody manufacturing in Pichia

Overview

Host creation

USP

YIELD QUALITY

Design of Experiments (DoE) based Process Development

Case studies

DSP

Analytics

www.ablynx.com

Process flow during Nanobody manufacture: up-scaling & increased control of product purity

R&D

Shake flask

Clone selection

based on

expression yield

& potency

R&D –

Host creation

Pichia based

clone selection

Yield & potency

USP

development

Fermentation

Expression > 1 g/L

Up-scaling: 2L 100L

Maximize yield &

purity

cGMP

manufacture

> 1500 L scale

Compliant to strict

regulatory guidelines &

product specifications

ABLYNX

Generation product for

clinical studies

CMO

DSP

development

Purification

Formulation

Maximize purity

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In-house large scale production of ~300 g Nanobody for

toxicological studies

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Nanobody manufacture from pipeline to clinic: short development timelines

Master

Cell Bank

Implementation and validation of analytical

methods in QC-GMP lab

Formulation buffer dev

Q1 Q2 Q3 Q4 Q5 Q6

Non GMP Stability trials

GMP manufacturing of

Drug Product

GMP Stability trials shelf-life / expiry date

Tasks at Ablynx

cGMP Tasks USP & DSP

development

Analytical

method dev

Up-scaling

(100L)

Host

creation

IND Single Dose

Tox study

Repeated Dose

Tox study

Selection

lead Tox DS IND

filing

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Nanobody expression platform

In-house production platform for Nanobodies based on Pichia pastoris

A typical Pichia

production

yields > 90 - 95%

pure Nanobody

Clarified Harvest

ALX-0141

E.coli Pichia

Pichia’s cell free

harvest equals a

capture step versus

clarified E. coli broth

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Pichia pastoris expression platform: Secretion of soluble Nanobody into the medium

Genomic integration single cross over event, homologous recombination (~90% efficient !),

Multiple insertions of the expression vector Higher expressions

n

Expression

vector

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Copy# 1 2

Nanobody A

Copy# 1 2 6

Nanobody B

Copy# 1 2 3 4 5

Nanobody C

correlation

Copy# 1 3 4

Nanobody D

Inverse

correlation Copy# 1 5

Nanobody E

For most Nanobodies a

positive correlation between

expression levels and copy

numbers is observed

(and preferred)

Pichia pastoris expression platform: Effect of copy number on expression level

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- Expression feasibility study

@shake flask level

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PRV

Guaranteed high titers and good quality

Product related variants

Stability

Formatting (linker length,

building blocks,...)

- Generic fermentation conditions

- >1 g/L cell free yield required

Pre-development: Investigation of manufacturability of Nanobody

lead candidates under generic conditions

- Generic purification

Nanobody Discovery

& formatting

Host

Creation

USP

DSP Analytics

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- Expression feasibility study

@shake flask level

- Strain selection (NRRLY-11430, X33,

KM71h, SMD1168H)

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Obtaining high titers and good quality

Product related variants

Stability

- Media screening (ABLY1- 55...)

- Parameter optimisation by DOE

Process Development: Crucial interactions between Host creation,

USP and DSP, supported by Analytics data about product related

variants

- Resin screening

- Parameter optimisation by DOE

Host

Creation

USP

DSP

Analytics

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UpStream Process Development Nanobody production via an optimal fermentation process

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2L

100L

→ Reproducible and scalable process

- High expression yield (>1g/L)

- High quality (low amount of product

related variants)

- Stable clarified fermentation broth

10L

USP fermentation optimization

→ Controlled production

(Temp, pH, Feed, DO )

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USP generic HCD Pichia pastoris fermentation

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Upstream Process Development

DoE to evaluate different fermentation parameters (2L scale)

growth medium, pH, temperature, methanol feed rate, pO2, …

Maximize product purity

Minimize degradation/product-

related variants

Maximize product titer

>1g/L

IPC/IPM

by

RP-HPLC

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YIELD QUALITY

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PRV Impurities

Product Related Variants versus Impurities

PRV (Potency = Active Drug)

• missing S-S

• O-glycosylation

• carbamylation

• leader sequence

• chemical degradants

• …

set specification in line with

observed process variability

preferably avoid @USP

Impurities (potency reduced or

lost)

• DNA

• HCP

• endotoxin

• antifoam, metals,

• degradation fragments

• …

set tight specifications (for some

EP/USP spec) in line with

anticipated dosing regimen

avoid @USP to levels below

specification and remove at DSP

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Preferred set-up: DoE Parameters

• % complex substrate in medium/feeds

• induction pH, temperature, DO

• MeOH feed rate

• ...

Responses

• expression yield

• proteolytic degradation (kinetics of

proteases and influenced by T, pH, ...)

• formation of Product Related Variants

- carbamylation

- unprocessed leader sequence

- chemical degradants

- …

Evaluation of

• main effects (pH, T, ..)

• interactions

Define optimal parameters

• maximize expression

• minimize PRVs

Interest of DSP

Upstream Process Development Optimization fermentation parameters via DoE

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Upstream Process Development Case-study: ALX-0141 (anti-RANKL)

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ALX-0141

• RankL key driver of bone resorption

• format: trivalent Nanobody, bispecific

two anti-RankL Nanobodies

one anti-HSA Nanobody (HLE)

• highly potent inhibition of target

• sub-cutaneous administration

• potential for multiple indications:

- osteoporosis

- cancer related bone loss

- rheumatoid arthritis

Manufacturing in Pichia pastoris

HLE

ALX-0141 – ~3.5 years

RANKL Ph. I

Fast Follower Program

July 2006: immunization of llamas

End 2009: initiated Phase I

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ALX-0141 secreted into the medium

Yield of 2 – 2.2 g/L in cell-free medium with low degradation after USP optimization

Scalable and reproducible process: 2L → 100L → 1000L scale

High yield, but what about purity…

Before USP optimization

Time of induction

SDS- PAGE of cell free medium during expression

After USP optimization

Time of induction

SDS- PAGE of cell free medium during expression SDS- PAGE of cell free medium during expression

Upstream Process Development Case-study: ALX-0141 (anti-RANKL)

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__SH HS__

10% of ALX-0141 with

missing canonical S-S bond

(+2 Da)

An unpaired cysteine variant (missing S-S bond) was observed (RP-HPLC analysis)

Removal at DSP is not efficient

A correlation between the observed titer and the % of missing S-S bound during

expression, i.e. the higher the titer the higher the % variant in the material

ALX-0141 after

incubation with

CuSO4

0%

___S......S__

_

Upstream Process Development Case-study: ALX-0141 (anti-RANKL)

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Anti-RSV Nanobody (ALX-0171) – first inhaled Nanobody

Mar 2010

Pre-clinical candidate

selected, ALX-0171

Initiation of pre-clinical

activities

H2 2011

Start Phase I

Project start and

immunizations initiated

Nov 2007

Successful generation of

trivalent functional Nanobody

Potent antiviral activity against

RSV

Feb 2009

Proof-of-concept for

nebulization and in vivo

antiviral activity

Oct 2009

anti-RSV

Nanobody

anti-RSV

Nanobody

anti-RSV

Nanobody

ALX-0171

42kD

Upstream Process Development Case-study: ALX-0171 (anti-RSV)

RSV: respiratory syncytial virus

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DOE_run 1

DOE_run 2

DOE_run 3

DOE_run 4

DOE_run 5

DOE_run 6

DOE_run 7

DOE_run 8

DOE_run 9

DOE_run10

Pre-peak 1

unprocessed

alpha mating

secretion peptide

Degradation

products

Post-peak 1

Carbamylated

variant

Post-peak 2

Unpaired cysteine

variants

Optimization of fermentation parameters via DoE set-up

Upstream Process Development Case-study: ALX-0171 (anti-RSV)

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Expression yield: significant effect of temperature, pH, MeOH feed and complex substrate

• high temperature and high pH promotes high yield, increasing MeOH feed lowers yield

• complex substrate influences expression yield & robustness of the process

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Degradation (pre-peaks): significant effect of induction temperature

• high temperature leads to higher proteolytic degradation

Carbamylation: significant effect of pH

• increases with higher pH

Unpaired cysteine variants: significant effect of induction temperature

• higher temperature reduces these variants, however, unpaired cysteine variants decrease if

induction times increases, possibly spontaneous oxidation

pH during

induction

Expression yield (g/L cell free medium)

Complex substrate 1 Complex substrate 2

pH 6.0 5.2 g/L 9.4 g/L

pH 6.2 6.3 g/L 10.1 g/L

pH 6.4 6.4 g/L 10.1 g/L

pH 6.6 9.3 g/L 11.3 g/L

Upstream Process Development Case-study: ALX-0171 (anti-RSV)

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Upstream Process Development Case-study: Nanobody X

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Clone A

Clone B

Importance of

clone screening

Final fermentation

process: a

compromise

between yield,

quality and stability

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Upstream Process Development Case-study: Nanobody X

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Optimal conditions

yield/quality/stability

Higher yield, but

decreased product

quality/stability

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DSP Development (DOE driven)

Locked DSP in 3 months time, ~100 chromatography runs

low in Product Related Variants (e.g. degradation products)

low in Process Related Variants (e.g. HCP, DNA …)

high yield (>60% recovery from USP)

low Cost of Goods (esp. resin cost)

CMC activities DownStream Processing

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1ml

6L

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Traditional DSP for Nanobodies

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•Capture Step Method

HCP/DNA removal

Volume reduction HIC, HCIC or CEX

• Intermediate Step Anion/Mixed mode

DNA/ HCP Reduction (Column/Filter in negative mode)

Aggregate removal

•Polishing Step Ion

Degradation removal Exchange

•UF/DF step

Formulation step Ultra Filtration

•Final Filtration (0.2 mm) Sterile Filtration

•Filling of DS

+/- Tween

DS: 10 150 mg/ml Total Product Yield

>60%

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Analytical methods are crucial for

Formulation/Stability

trials

In process monitoring

of purity and quantity

Characterization variants

In-use stability /

compatibility

Release testing

Shelf-life /

expiry date

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GMP

QC unit

Process

Development

Analyses/process

350 RPC

300 SEC

200 HCP

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CMC process flow

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Clone selection & RCB generation

Process Development (USP & DSP) Analytics

Analytical Package and Formulation Development

Confirmation and Pilot run at 100L scale

Ablynx cGMP QC Unit

cGMP run outsourced to CMO

(~1000L scale)

Fill & Finish

DS → DP

4-5 months

Tech

Transfer

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Questions?

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