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
Nanobodies® Inspired by nature
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.
www.ablynx.com
2
Outline
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
3
Company highlights
Corporate
•
•
•
•
Drug discovery and development company - Ghent, Belgium
NYSE Euronext Brussels (ABLX)
49M shares outstanding (52M fully diluted)
280 employees
Technology
•
•
•
Pioneer in next generation biologics – Nanobodies®
>500 granted and pending patents
Products
•
•
•
•
~30 programmes – seven in clinical development
Two clinical proof-of-concepts
>800 healthy volunteers and patients treated with Nanobodies
Partners
•
•
•
AbbVie, Boehringer Ingelheim, Eddingpharm, Merck & Co,
Merck Serono and Novartis
>€300M in non-dilutive cash received to date
Financials
•
•
~ €200M in cash estimated at 31st December 2013
~ €20-25M net cash burn estimated for the full year 2013
www.ablynx.com
4
Nanobodies – proven single variable domain approach
Camelidae family has both forms
CH1
CL
VH
VL
VHH
VHH
CH2
CH2
CH3
CH3
Ablynx’s Nanobody®
• Small (1/10 size of a mAb)
• Flexible formatting
• Highly potent, robust and stable
Conventional antibody
Heavy-chain antibody
•
Heavy and light chains
•
Only heavy chains
•
Both chains required for antigen
binding and stability
•
Full antigen binding capacity
and very stable
•
Large size and relatively low
formatting flexibility
•
Administered through injection
www.ablynx.com
• Broad target applicability
• Multiple administration routes
• Ease of manufacture
• Speed of discovery
5
Nanobodies – uniqueness and competitive advantages
Broad target applicability, including challenging targets such
as GPCRs and ion channels
Flexible formatting: multivalent, multi-specific, bi-paratopic
Nanobodies
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)
www.ablynx.com
6
Nanobody discovery process
Immunize llama
with antigen
Draw blood 6–12
weeks later
Conventional
antibodies
VHH
CH2
CH3
Manufacture in
micro-organisms
Clinical trials
www.ablynx.com
plus half-life
extension
(HLE)
VHH
Ablynx’s
Nanobody®
Select Nanobodies
of interest
Format Nanobody to achieve
desired properties
7
Programmes in Phase I and II clinical development
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
www.ablynx.com
8
Outline
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
9
Process flow during Nanobody manufacture:
up-scaling & increased control of product purity
R&D –
Host creation
R&D
Shake flask
Pichia based
clone selection
USP
development
DSP
development
Fermentation
Expression > 1 g/L
Up-scaling: 2L  100L
Purification
Formulation
Clone selection
based on
expression
Yield & potency yield  Maximize yield &
purity
& potency
www.ablynx.com
Compliant to strict
regulatory guidelines &
product specifications
 Maximize purity
In-house large scale production of ~300 g Nanobody for
toxicological studies
ABLYNX
cGMP
manufacture
> 1500 L scale
Generation product for
clinical studies
CMO
10
Nanobody manufacture from pipeline to clinic:
short development timelines
Selection
lead
IND
filing
Tox DS
Q1
Q2
Host
creation
Q3
Q4
Master
Cell Bank
Q6
Tasks at Ablynx
cGMP Tasks
USP & DSP
development
Up-scaling
(100L)
Single Dose
Tox study
Analytical
method dev
Q5
GMP manufacturing of
Drug Product
Repeated Dose
Tox study
IND
Implementation and validation of analytical
methods in QC-GMP lab
Formulation buffer dev
Non GMP Stability trials
www.ablynx.com
GMP Stability trials  shelf-life / expiry date
11
Nanobody expression platform
In-house production platform for Nanobodies based on Pichia pastoris
Clarified Harvest
E.coli Pichia
A typical Pichia
production
yields > 90 - 95%
pure Nanobody
ALX-0141
Pichia’s cell free
harvest equals a
capture step versus
clarified E. coli broth
www.ablynx.com
12
Pichia pastoris expression platform:
Secretion of soluble Nanobody into the medium
Expression
vector
n
Genomic integration single cross over event, homologous recombination (~90% efficient !),
Multiple insertions of the expression vector  Higher expressions
www.ablynx.com
13
Pichia pastoris expression platform:
Effect of copy number on expression level
Copy#
1
2
2
6
Nanobody A
Copy#
correlation
1
Nanobody B
Copy#
1
2
3
4
5
Nanobody C
Copy#
1
3
4
1
5
For most Nanobodies a
positive correlation between
expression levels and copy
numbers is observed
(and preferred)
Nanobody D
Inverse
correlation
Copy#
Nanobody E
www.ablynx.com
14
Guaranteed high titers and good quality
Pre-development: Investigation of manufacturability of Nanobody
lead candidates under generic conditions
Nanobody Discovery
& formattingPRV
Host
Creation
- Expression feasibility study
@shake flask level
Formatting (linker length,
building blocks,...)
USP
Analytics
DSP
- Generic fermentation conditions
- >1 g/L cell free yield required
- Generic purification
Product related variants
Stability
www.ablynx.com
15
Obtaining high titers and good quality
Process Development: Crucial interactions between Host creation,
USP and DSP, supported by Analytics data about product related
variants
Host
Creation
- Expression feasibility study
@shake flask level
- Strain selection (NRRLY-11430, X33,
KM71h, SMD1168H)
USP
- Media screening (ABLY1- 55...)
- Parameter optimisation by DOE
DSP
- Resin screening
- Parameter optimisation by DOE
Analytics
Product related variants
Stability
www.ablynx.com
16
UpStream Process Development
Nanobody production via an optimal fermentation process
USP fermentation optimization
→ Controlled production
(Temp, pH, Feed, DO )
2L
100L
10L
→ Reproducible and scalable process
- High expression yield (>1g/L)
- High quality (low amount of product
related variants)
- Stable clarified fermentation broth
www.ablynx.com
17
USP generic HCD Pichia pastoris fermentation
www.ablynx.com
18
Upstream Process Development
DoE to evaluate different
fermentation parameters (2L scale)
growth medium, pH, temperature, methanol
feed rate, pO2, …
YIELD
IPC/IPM
by
RP-HPLC
Maximize product
titer
>1g/L
www.ablynx.com
QUALITY
Maximize product
purity
Minimize
degradation/productrelated variants
19
Product Related Variants versus Impurities
PRV (Potency = Active Drug)
•
•
•
•
•
•
PRV
missing S-S
O-glycosylation
carbamylation
leader sequence
chemical degradants
…
 set specification in line with
observed process variability
Impurities (potency reduced or
Impurities
lost)
•
•
•
•
•
•
DNA
HCP
endotoxin
antifoam, metals,
degradation fragments
…
 set tight specifications (for some
EP/USP spec) in line with
anticipated dosing regimen
 preferably avoid @USP
avoid @USP to levels below
specification and remove at DSP
www.ablynx.com
20
Upstream Process Development
Optimization fermentation parameters via 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
- …
www.ablynx.com
Preferred set-up: DoE
Evaluation of
• main effects (pH, T, ..)
• interactions
Define optimal parameters
• maximize expression
• minimize PRVs
Interest of DSP
21
Upstream Process Development
Case-study: ALX-0141 (anti-RANKL)
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
HLE
RANKL
Manufacturing in Pichia pastoris
www.ablynx.com
ALX-0141 – ~3.5 years
Ph. I
Fast Follower Program
July 2006: immunization of llamas
End 2009: initiated Phase I
22
Upstream Process Development
Case-study: ALX-0141 (anti-RANKL)
ALX-0141 secreted into the medium
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
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…
www.ablynx.com
23
Upstream Process Development
Case-study: ALX-0141 (anti-RANKL)
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
__SH
HS__
___S......S__
_
10% of ALX-0141 with
missing
canonical
S-S bond
ALX-0141
after
(+2 Da)with
incubation
CuSO4
0%
www.ablynx.com
24
Upstream Process Development
Case-study: ALX-0171 (anti-RSV)
Anti-RSV Nanobody (ALX-0171) – first inhaled Nanobody
Mar 2010
Pre-clinical candidate
selected, ALX-0171
Feb 2009
Successful generation of
trivalent functional Nanobody
Initiation of pre-clinical
activities
Potent antiviral activity against
RSV
ALX-0171
42kD
anti-RSV
Nanobody
anti-RSV
Nanobody
Nov 2007
Project start and
immunizations initiated
anti-RSV
Nanobody
Oct 2009
Proof-of-concept for
nebulization and in vivo
antiviral activity
H2 2011
Start Phase I
RSV: respiratory syncytial virus
www.ablynx.com
25
Upstream Process Development
Case-study: ALX-0171 (anti-RSV)
Optimization of fermentation parameters via DoE set-up
Post-peak 1
Carbamylated
variant
DOE_run 1
DOE_run 2
DOE_run 3
DOE_run 4
DOE_run 5
DOE_run 6
DOE_run 7
Pre-peak 1
unprocessed
alpha mating
secretion peptide
DOE_run 8
DOE_run 9
DOE_run10
Post-peak 2
Unpaired cysteine
variants
Degradation
products
www.ablynx.com
26
Upstream Process Development
Case-study: ALX-0171 (anti-RSV)
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
Expression yield (g/L cell free medium)
pH during
induction
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
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
www.ablynx.com
27
Upstream Process Development
Case-study: Nanobody X
Importance of
clone screening
Clone B
Final fermentation
process: a
compromise
between yield,
quality and stability
Clone A
www.ablynx.com
28
Upstream Process Development
Case-study: Nanobody X
Optimal conditions
yield/quality/stability
Higher yield, but
decreased product
quality/stability
www.ablynx.com
29
CMC activities
DownStream Processing
DSP Development (DOE driven)
1ml
6L
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)
www.ablynx.com
30
Traditional DSP for Nanobodies
• Capture Step
Method
HCP/DNA removal
Volume reduction
HIC, HCIC or CEX
• Intermediate Step
Anion/Mixed mode
DNA/ HCP Reduction
Aggregate removal
(Column/Filter in negative mode)
• Polishing Step
Ion
Exchange
Degradation removal
• UF/DF step
Formulation step
Ultra Filtration
+/- Tween
• Final Filtration (0.2 mm)
• Filling of DS
Total Product Yield
>60%
www.ablynx.com
Sterile Filtration
DS: 10  150 mg/ml
31
Analytical methods are crucial for
GMP
QC unit
Release
testing
Formulation/
Stability
trials
Shelf-life /
expiry date
In process
monitoring
of purity and
quantity
Process
Development
In-use
stability /
compatibility
www.ablynx.com
Characterization
variants
Analyses/process
350 RPC
300 SEC
200 HCP
32
CMC process flow
Clone selection & RCB generation
Process Development (USP & DSP)  Analytics
4-5 months
Analytical Package and Formulation Development
Confirmation and Pilot run at 100L scale
Tech
Transfer
Ablynx cGMP QC Unit
cGMP run outsourced to CMO
(~1000L scale)
www.ablynx.com
Fill & Finish
DS → DP
22
Questions?
www.ablynx.com
34