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Nanobodies ® -Inspired by nature
Strategies and Challenges in Analytical Testing of Nanobody-Based Multispecifics
Ann Brigé, Section Head CMC Analytics
5th World Bispecific Antibody Summit
September 23-25, 2014 – Boston
www.ablynx.com
Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
2
www.ablynx.com
Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
3
www.ablynx.com
Corporate snapshot
• Drug discovery and development company in Ghent, Belgium• >300 employees
4
Corporate
Technology
Partners
Products
• Pioneer in next generation biologics – Nanobodies®
• >500 granted and pending patents
• >30 programmes – six in clinical development• Three clinical proof-of-concepts (POC)• >900 healthy volunteers and patients treated with Nanobodies
• AbbVie, Boehringer Ingelheim, Eddingpharm, Merck & Co,Merck Serono and Novartis
Financials• €196M in cash at 30th June 2014• Raised €41.7M through private placement (3rd July 2014)
www.ablynx.com
Camelid heavy-chain only antibodies are stable and fully functional
Nanobodies represent the next generation of antibody-derived biologics
Nanobodies – derived from heavy-chain only antibodies
Conventional antibodies
Heavy chain only antibodies
Ablynx’s Nanobody• unique formatting flexibility (up to
penta-valent)
• speed and ease of generating multi-specifics
• nano- to picomolar affinities
• favourable biophysical properties (Tm, solubility, viscosity)
• tackling intractable targets
• multiple administration routes
• manufacturing in microbial cells
CH2
CH3
CH1
CL
VL
VH 12-15kDa
CH2
CH3
VHH
VHH
5
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Custom therapeutics... which go beyond antibodies and fragments....
Flexible formatting with tailored half-life (hours to weeks):multivalent, multispecific, biparatopic Nanobodies in the clinic
www.ablynx.com
Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
7
www.ablynx.com 8
Analytical testing of biopharmaceuticals
Formulation studies
In process monitoring /
control
Release testing
Stability testing
Characterization
In-use stability / compatibility
CONSISTENT
ACTIVE
SAFE
Shelf-life
Define formula & dosage form
Process development & manufacturing
Pre-defined acceptance criteria
Product understanding
Clinical admini-stration studies
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Analytical testing of biopharmaceuticals
9
Long-term involvement from lead identification to commercial & beyond
The challenge is to fulfil all regulatory requirements, which become more stringent as development advances
Pre-clinicalPre-clinical Phase IPhase I Phase IIPhase II Phase IIIPhase III
• Initial characterization• Scientifically sound
methods• Preliminary specifications• Preliminary stability
• Full characterization (ICH Q6B), including all molecular variants
• Validated test methods• Long-term stability testing (>3 years)• Final specifications• Expiration date / shelf-life • Comparability study, if required for
process change
www.ablynx.com
Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
10
www.ablynx.com
Case study bispecific lead candidate - CMC development
11
USP & DSP 100L pilot scale, scaleable to >1500L
Formulation Liquid, 150 mg/mL
AnalyticsKey methods: potency, SE-HPLC, RP-UHPLC & cIEF
Test procedures for batch release & stability testing
Analysis in-process samplesCharacterization
Target 2
Unformed S-S bridgeDegradation
Distinct HMW species
Analytics
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ICH Q6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
12
Potency
Identity
Purity - product-related impurities
Purity - process-related impurities
Quantity
General/additional tests
Appearance
Product-specific methods
TO BE DEVELOPED
BIVALENT
BISPECIFIC
= / ≠
BIVALENT
BISPECIFIC
=
Generic/compendial methods (USP/Ph.Eur)
IN PLACE
HCP, DNA, endotoxin, ...
Protein content (OD280)
pH, osmolality, sub-visible particles, sterility, bioburden, container
closure integrity, ...
Appearance testing
RELEASE/STABILITY
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ICH Q2 (R1) Method development and validation
Demonstrate that the method is suitable for its intended purpose
Methods should be stability-indicating • to monitor any form of modification or degradation during manufacturing, at
release & during stability
• include samples from eg forced degradation & stress studies during development
Validation parameters are method-dependent, specificity required
Validation parameters IdentityPurity*
Assay Content/potencyQuantitative
test*Limit test**
Accuracy X XPrecision: Repeatibility X XPrecision: Intermediate pr. X XSpecificity X X X XDetection limit (X) XQuantitation limit XLinearity X XRange X X
* For the active product or other selected components in DS/DP; ** For the control of impurities
13
RELEASE/STABILITY
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ICH Q6B Potency
“Potency is the quantitative measure of biological activity based on the attribute of the product which is linked to the relevant biological properties. Biological activity describes the specific ability or capacity of a product to achieve a defined biological effect.”
Relevant assays can have different format• animal-based bioassays (In-vivo assay)
• cell culture-based bioassays (In-vitro assay)
• biochemical assays (binding assay), eg ELISA-based & SPR
14
RELEASE/STABILITY
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1 target = 1 functionality1 assay to be developed
1 target = 1 functionality1 assay to be developed
2 targets = 2 functionalities2 assays to be developed
Approaches for potency assay development -demonstration of specificity
15
BIVALENTBISPECIFIC MONOVALENT
Unrelated Nanobody to demonstrate specificity
(same format)
Engineered mono-functional variants & unrelated
Nanobody to demonstrate avidity & specificity
Engineered mono-specific variants & unrelated
Nanobody (same format) to demonstrate specificity
RELEASE/STABILITY
OD
45
0 n
m MonoBi
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Potency assay for target 1
Inhibition assay
Example: discrimination from less potent variant
16
Less potent variant0.01 0.1 1 10 100 1000
0.0
0.5
1.0
1.5
2.0lead anti-IgE Nb
less potent variant
monovalent anti-IgE Nb
BN
CI
NB (nM)
X X
RELEASE/STABILITY
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Potency assay for target 2
Binding assay
Example: discrimination from monospecific variant (unrelated Nanobody for target 2) and unrelated Nanobody for both targets
17
Target 2
Target 2X
XTarget 2
RELEASE/STABILITY
OD
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ICH Q6B Identity
“Identity test(s) should be highly specific for the drug substance and should be based on unique aspects of its molecular structure and/or other specific properties. More than one test may be necessary to establish identity. The identity test(s) can be qualitative in nature.”
MW (SDS-PAGE)
pI (cIEF)
Activity (Potency assay/s)
18
kDa
RefDifference between MW of test sample
and Reference Standard ≤ 2 kDa
RELEASE/STABILITY
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ICH Q6B Identity
“Identity test(s) should be highly specific for the drug substance and should be based on unique aspects of its molecular structure and/or other specific properties. More than one test may be necessary to establish identity. The identity test(s) can be qualitative in nature.”
MW (SDS-PAGE)
pI (cIEF)
Activity (Potency assay/s)
19
4.3 5.7 9.1
pI 5.6 ± 0.2
RELEASE/STABILITY
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ICH Q6B Identity
“Identity test(s) should be highly specific for the drug substance and should be based on unique aspects of its molecular structure and/or other specific properties. More than one test may be necessary to establish identity. The identity test(s) can be qualitative in nature.”
MW (SDS-PAGE)
pI (cIEF)
Activity (Potency assay/s)• Response comparable to reference standard → specific ability or capacity
of a product to achieve a defined biological effect
20
RELEASE/STABILITY
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ICH Q6B Purity
“The absolute purity of biotechnological and biological products is difficult to determine and the results are method-dependent. Consequently, the purity of the drug substance is usually estimated by a combination of methods . The choice and optimisation of analytical procedures should focus on the separation of the desired product from product-related substances and from impurities .”
Examples obtained during analysis of in-process analysis and characterization studies (next slides)
21
Method Separation Reporting at release(Non)-reducing SDS-PAGE
LMW & HMW variants/impurities % main band
SE-HPLC LMW & HMW variants/impurities % main peak, % HMW
RP-UHPLC Variants with changed hydrophobicity % main peak
cIEF Charge variants % main peak
RELEASE/STABILITY
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Purity analysis of in-process samples
Importance during Process development & manufacturing
(purity assessment and impurity profiling)
Increase process and product understanding
22
Upstream & Downstream Process development
Evaluation of different process parameters
Maximize product purityProcess- & product-
related impurities
Maximize product titer &
recovery
Analysis in-process samples
IN PROCESS
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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00-10.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0mAU
min
21
Fermentation sample Fermentation sample after Cu addition
Example 1: development data USP (Pichia pastoris)
Titer: 12 g/L cell free medium
Purity by RP-UHPLC • product variant with unformed disulphide bond (+2Da in LC-MS)
• identified in second building block (degradation fragment)
• formation disuphide bond can be induced by addition of Cu-sulphate
Outcome in-process analysis: optimization fermentation conditions by adding Cu prior to harvest to increase product purity
23
RP-UHPLC
-SH HS-
-S-S--S-S-
-S-S-
-S-S--S-S-
-SH HS-
IN PROCESS
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Analysis in-process samples to:
Define process parameters
Demonstrate removal/clearance of process-and product-related impurities
Demonstrate stability of intermediate samples (hold-time studies)
Example 2: development data DSP
HARVEST
CAPTURE
POLISH
UF/DF
STERILE FILTRATION
24
IN PROCESS
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Hold-time studies on process intermediates• formation of product-related variant
• detected by RP-UHPLC and cIEF, not by SDS-PAGE and SE-HPLC
• identification by mass spectrometry (LC-MS):
- degradation fragment without 5 N-terminal amino acids
• outcome in-process analysis: optimization capture and polish step elution conditions & collection criteria to remove residual proteases
Further characterization product-related variant
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0-10
0
10
20
30
40
50
60
70
80
min
21
Example 2: development data DSP
25
Non-optimized process step -5AA (%)
Capture step
Eluate 0.6Eluate, 3d 2-8°C 3.1
Eluate, 3d RT 8.3
Polish step
Eluate <0.1
Eluate, 3d 2-8°C 0.4
Eluate, 3d RT 1.4
IN PROCESS
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Characterization
Determination of physicochemical properties, biological activity, immunochemical properties, purity and impurities (quality attributes)
Identification & characterization of product-related variants
26
CHARACTERIZATION
Product-related substance
Same potency, efficacy, safety as desired product
Preferentially to be removed during USP/DSP
Set specification in line with observed process variability
Product-related impurity
Not the same potency, efficacy, safety as desired product
To be removed during USP/DSP to levels below
specification
Set specification to control level of impurities
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Example 1: characterization -5AA fragment observed during process development
Test samples• purified -5AA fragment (90% pure)
• recombinantly produced as truncated version of bispecific (100% pure)
Unexpected loss in potency for target 1 (5 N-terminal amino acids are not part of any of the CDRs) → impurity
27
= recombinant variant
= purified fragment
CHARACTERIZATION
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Example 1
Destabilization predicted by structural modelling: N-terminal residues stabilized by interactions with CDR1 and CDR3/FR4
28
CHARACTERIZATION
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Example 1
Thermal unfolding experiment using DSC
Two building blocks unfold separately in -5AA variant → different structural conformation & destabilization confirmed
29
30 40 50 60 70 80 90-0.000025
-0.000020
-0.000015
-0.000010
-0.000005
0.000000
0.000005
0.000010
0.000015
0.000020
0.000025
0.000030
0.000035
ALX-0962-RS-01
Cp(
cal/o C
)
Temperature (oC)
Bispecific (intact)
30 40 50 60 70 80 90-0.000025
-0.000020
-0.000015
-0.000010
-0.000005
0.000000
0.000005
0.000010 IgE126 F(126)130415/1
Cp(
cal/o
C)
Temperature (oC)
30 40 50 60 70 80 90
-0.000020
-0.000015
-0.000010
-0.000005
0.000000
0.000005
0.000010
0.000015
0.000020 IgE00122 Q Sepharose HP cycle b P1/130412/1 pre-peak 2
Cp(
cal/o C
)
Temperature (oC)
Recombinant variant (-5AA) Purified fragment (-5AA)
64°C 67°C 68°C
50°C50°C
CHARACTERIZATION
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Example 2: characterization during formulation & preliminary stability studies
Appearance of distinct HMW species after prolonged temperature stress
MW determination by SE-HPLC-MALS: dimer
30
CHARACTERIZATION
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0-20
25
50
75
120 3 - IgE00122 REF [modified by jstorms] SEMA0096X0015D MWD1_A__Sig_280_4_Ref_600_100mAU
min
Abs
orba
nce
[mA
U]
Retention T ime [min]
321
ReferenceControl (unstressed)Stressed
SE-HPLC-UV25.3 kDa - monomer
51.3 kDadimer
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Example 2
31
NB (nM)
Relative potency (CI)Reference /Control (unstressed) 1.044 (0.927-1.177)Dimer fraction 0.454 (0.360-0.572)Monomer fraction 0.947 (0.839-1.069)Stressed 0.844 (0.730-0.975)Monomer fraction (2) 0.937 (0.826-1.063)
CHARACTERIZATION
Inhibition assay target 1
Target 2Target 2
Target 2
Relative potency
FractionInhibition assay
target 1Binding assay
target 2
dimer � �
monomer = =
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Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
32
www.ablynx.com
Conclusion
Nanobodies are extremely modular, allowing for full control over valency, multispecificity and half-life
Analytical methods play a crucial role in all stages during development of pharmaceutical drugs
Establishing the analytical methods for bi/multi-specifics requires different approaches compared to mono-specifics
Availability of stability-indicating, product-specific methods is very valuable already in the early stages of development
33
www.ablynx.com
Outline
Introduction to Ablynx & Nanobody platform
Analytical testing of biopharmaceuticals
Case study bispecific Nanobody
• test procedures for release & stability testing
• analysis in-process samples
• characterization
Conclusion
Acknowledgments
34
www.ablynx.com
Acknowledgments
CMC Analytics
Veronique De Brabandere
An Cerdobbel
Julie Storms
Tom Merchiers
Nathalie Moens
Saskia De Keyser
Mathijs Clauwers
Lutgarde Brewaeys
Mabelle Meersseman
Annelies Clottens
Liesbeth Lambert
Niels Meersman
Justien Maes
Liese Vandevyvere
35
CMC Process Development
Peter Casteels
Manu De Groeve
Ellen Van Horen
Anneleen Devos
Marianne Habets
Willem Van de Velde
Sindi De Vrieze
Bieke Vlieghe
Ruben Christiaen
Wouter Martens
Glenn Goegebeur
Rudy Cocquyt
... and many others !
Nanobodies ® -Inspired by nature
Strategies and Challenges in Analytical Testing of Nanobody-Based Multispecifics
Ann Brigé, Section Head CMC Analytics
5th World Bispecific Antibody Summit
September 23-25, 2014 – Boston
