Pharma&Biotech
Fast-Track Expression and Production of Next-Generation Therapeutics with Lonza’s XS™ Pichia Platform
Christoph Kiziak, Microbial Research & Technology
18 January 2017 Forward-Looking Statements
Certain matters discussed in this presentation may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. Investors are cautioned that all forward-looking statements involve risks and uncertainty and are qualified in their entirety. The actual results may differ materially in the future from the forward- looking statements included in this presentation due to various factors. Furthermore, except as otherwise required by law, Lonza Group Ltd disclaims any intention or obligation to update the statements contained in this presentation.
2 Feb-19 Overview
. XS™ Microbial Expression Technologies . Why a new promoter for Pichia? . Introduction to the Glucose Regulated Platform and the G1-3 Promoter . Clone Screening in Microtiter Format . Fermentation Regimes / Case Studies . Classical feed regimes: (1a) Simple / (1b) Conventional . Advanced model-based feed designs: (2a) Speed + (2b) Titer . Summary . Questions
3 Feb-19 Biologic Pipelines are Evolving
. Novel disease targets and biological mechanisms Bispecific Expression require new molecule scaffolds
Either Microbial . There is demand for target therapeutics with 25% 28% improved efficacy . Pipelines moving from standard format MAbs to new bi- and multispecific formats Mammalian . New molecular formats provide patient benefit 47% and demonstrable clinical cost effectiveness Citeline Analysis, Feb 2016 . Expression challenges – sufficient quantity at required quality . No expression system is currently addressing all requirements . Molecules are expressed in either mammalian or microbial systems . XS Pichia as a valuable alternative to bacterial or mammalian expression
4 Feb-19 Multiple Host Options for Different Products
Escherichia coli Pichia pastoris Bacillus subtilis
- Expresses >80% - Protein folding - Expresses and of commercial issues in E. coli secretes simple microbial products may be solved monomeric with Pichia proteins - Cytoplasmic and periplasmic - Secretes for - Lacks endotoxin production easier recovery - Produces and downstream aglycosylated - Produces processing protein aglycosylated - Lacks endotoxin proteins
5 Feb-19 The XS™ Toolbox – A Broad Portfolio
XS Technologies™
E. coli Pichia Bacillus
Sugar Depletion IPTG Glucose Strong Methanol GAP Sugar Auto Inducible Inducible Inducible Regulated Constitutive Inducible Constitutive Inducible Inducible
Host strains
Signal sequences
Integration sites
Helper factors
6 Feb-19 Why a New Promoter for Pichia?
Intro 1: Lonza Custom manufacturing
70 L – 15’000 L Microbial Pharma cGMP Production
Speed Robustness Reliability Processability
7 Feb-19 Titer Improvement Comes with Complexity
derepr. mixed glucose feed glycerol mutS sorbitol
AOX1 + mut μmax lysis
performance complexity
medium hypoxic EtOH GAP titer
8 Feb-19 New Lonza XS™ Pichia Systems Innovation versus Improvement
“The light bulb wasn’t invented AOX1 by continuously G1-3 Glucose improving the regulated candle…”
GAP CS1 Strong constitutive
9 Feb-19 XSTM Pichia Glucose-regulated Platform Taking Fermentation Needs into Account
C 7 strong and tightly regulated promoters identified
G1 as the most promising candidate for product formation BATCH FED-BATCH REPRESSION INDUCTION Glucose Transporter High affinity 1 Carbon excess Glucose limit GTH1 Further molecular biological Identification of improvements led to G1-3 regulated genes
s The whole set can be used for 60 G4 perfectly tuned expression of 7 promoters with 40 Activity G6 G3 similar regulation, G7 helper factors, heteromeric but different 20
relative eGFP expression [%] expression eGFP relative G1 induction pattern Relative 0 products, enzyme cascades etc.
0.001 0.01 0.1 1 10 100
-1 GlucoseGlucose [g [gL L ] -1]s 10 Feb-19 Clone Screening in Microtiter Format Simplifying Development
Glucose regulated G1-3 MetOH induced AOX1
MetOH spikes 120 120
100100
1193 –
8080
(%) (%)
2 2 60
2 2 60
pO pO
4040
Advances 33, 1177 et al., 2015, et 2015, al.,
2020 Looser Looser
0 0 From 0 0 2020 4040 60 60 80 80 Biotechnology processprocess time time (h) (h) *
Convenient addition of a glucose feed bead vs. MetOH spiking
Controlled and robust with high reproducibility, easy to measure
Short screening resembling a fed-batch fermentation process
11 Feb-19 High Purity from the Start Supernatants from Microtiter Plate Screening
Pichia MTP supernatants (undiluted) 1 2 3 4 5 6 7 8 9
4 domain multi-DARPin®
2 domain multi-DARPin®
Monomeric DARPin®
First non-purified
ESI-MS product of total MTP available supernatant 4-6 weeks of the monomeric DARPin® molecule after gene after ZipTipTM desalting delivery
12 Feb-19 Fermentation regimes
• XS™ Microbial Expression Technologies • Why a new promoter for Pichia? • Introduction to the Glucose Regulated Platform and the G1-3 Promoter • Clone Screening in Microtiter Format • Fermentation Regimes / Case Studies • Classical feed regimes: (1a) Simple / (1b) Conventional • Advanced model-based feed designs: (2a) Speed + (2b) Titer • Summary, Q&A
13 Feb-19 Fermentation Benchmarking Study G1-3 versus AOX1
Reporter molecule i-bodyTM provided by AdAlta Single domain antibody-like molecule of human origin Host strain Wild type mut+ without any helper factors Signal peptide a-mating factor Integration site AOX1 Minimal Screening 11 clones per construct, no GCN optimization Temperature 25°C Screening titer in MTPs G1-3: 77 mg/L (1x glucose feed bead) < AOX1: 102 mg/L (4x MetOH shots) Easy fed-batch like screening setup for G1-3 Solid performing strains with further optimization potential Typical MTP screening results for various proteins show similar expression levels for both systems 14 Feb-19 (1a) Basic Fermentation Simple Feed Regime
Glucose regulated G1-3 MetOH induced AOX1
Simple 2-step fermentation regime for G1-3 without addition of an inducer No toxic effects of MetOH and its metabolites
Robust fermentation setup for non explosion-proof plants
15 Feb-19 (1a) Basic Fermentation Simple Feed Regime
Glucose regulated G1-3 MetOH induced AOX1
Higher titer compared to AOX1 mut+ with less biomass formation Defined media without complex compounds Tightly repressed under carbon excess conditions in batch phase
16 Feb-19 (1b) Conventional Fermentation 2-step Feed Regime
1 1 2 pG1-3
pAOX1 (**)
pAOX1 1 Biomass (and product) formation
2 Product formation pGAP
* For comparison only, extracted from fermentation data ** From Looser et al., 2015, Biotechnology Advances 33, 1177–1193 17 Feb-19 (1b) Conventional Fermentation 2-step Feed Regime
Glucose regulated G1-3 MetOH induced AOX1
Wide performance range with high titer in reduced total fermentation time
Lower oxygen consumption and heat generation for disposable manufacturing
18 Feb-19 (1b) Conventional Fermentation 2-step Feed Regime
Glucose regulated G1-3 MetOH induced AOX1
Max. titer
G1-3 result AOX1 6.2 max. titer (g L-1) 3.6 Space time yield STY “performance of the process” 96 h reached at 125 h from t=0 to sampling time 31 highest STY (mg L-1 h-1) 21 Specific productivity qP -1 -1 “performance of the biomass” 2.0 highest qP (mg g h ) 0.6 between the last two sampling 129 cell density (g L-1) 109 points
80 x increase of titer (vs. MTP) 35 x 19 Feb-19 (2) Advanced Model-based Feed Designs
Max. Model Space Time selection Yield Experimental Add constraints Data e.g. maximum DCW
Choose Optimization objective of feed profile
Physicochemical parameter determination High Titer
Fermentation Data for Generation of Basic operating Model conditions like Experimental pH and temp. Early material from verification depending on Standard product Fermentation Final clone selection
20 Feb-19 (2a) Speed Fermentation Maximizes Space Time Yield
Feed regime for maximum Space Time Yield (STY) based on (product–specific) growth rate - productivity model
21 Feb-19 (2a) Speed Fermentation Maximizes Space Time Yield
Total fermentation time of 60h comparable to «bacterial» systems
High titer and short product retention time in the supernatant
Supernatant titer of > 5 g/L after 48 h at 25°C
22 Feb-19 (2a) Speed Fermentation Maximizes Space Time Yield
G1-3 G1-3 result AOX1 Speed Standard 6.4 6.2 max. titer [g L-1] 3.6 60 h 96 h reached at 125 h
58 31 highest STY [mg L-1 h-1] 21
-1 -1 2.3 2.0 highest qP [mg g h ] 0.6 139 g L-1 129 g L-1 cell density 109 g L-1
80 x 80 x increase of titer (vs. MTP) 35 x 23 Feb-19 Transferability and Robustness Production of an Antibody Mimetic
Three domain antibody mimetic molecule provided by Affibody Wild type host, Lonza SP2, Lonza MC integration site, 11 clones screened in MTP ► Minimal screening program, no GCN optimization
► 1 fermentation in total Biomass limit: 120 g/L, Max. total fermentation time of 48 h at 30°C
Direct implementation of Speed Fermentation regime without product specific adaptations delivered 3 g/L of product in 2 days of total fermentation time for a non-optimized strain
24 Feb-19 (2) Model-based Feed Designs High Titer and max. Space Time Yield
Reporter molecule DARPin® provided by Molecular Partners Engineered scaffold protein therapeutic derived from natural ankyrin repeat proteins
Wild type host, a-MF, Lonza MC integration site, 12 clones screened in MTP ► Minimal screening program, no GCN optimization
Parameter Model generation determination with 30°C, pH 6 2 fermentations 25 Feb-19 (2) Model-based Feed Design Comparing High Titer and Max Space Time Yield
Max. STY
High titer
Product Fermentation Titer [g L-1] STY [mg L-1 h-1] Feed time [h]
Objective 1.9 19.7 31 Model-based max STY DARPin® optimization Objective high titer 3.5 12.5 118 26 Feb-19 Process Variability Window for the G1-3 Fermentation
4 Objective function The G1-3 promoter High titer enables a customized 3
fermentation regime
] 1 based on the -
gL ProductionProduction PlantPlant/ / 2 requirements of the Process
full production limitations Titer [ Titer process and the Biomass separation 1 Maximum load specifications of the capture column production plant Batch cycle time 0 0 20 40 60 80 100 120 140 Feed time [h] Objective function Typical product accumulation of an AOX1 process Maximum assuming a similar final titer after 120h Space Time Yield 10h for glycerol feed and MetOH bolus 27 Feb-19 It’s not only about titer…
High cell viability with low cell lysis rate XSTM Pichia G1-3 speed fermentation Low level of host cell impurities (HCP) in of a DARPin® Pichia supernatant facilitates DSP Fermentation supernatants Purity [%] ≥ 70% 1:10 dilution undiluted
after induction Shorter fermentation time / product
7h
33h 15h 20h Marker 13h 13h retention time can reduce potential modifications of the product
Microscopic evaluation of Pichia culture broth at the end of a G1-3 fermentation
28 Feb-19 Summary XS™ Pichia G1-3
Simple screening High titers Robust and scalable, and straightforward in short total suitable for fermentation regimes fermentation time disposable systems
G1-3 Supernatant titer + 80% Total fermentation time
GLYCEROL GLUCOSE - 125% BATCH FED-BATCH
AOX1
GLYCEROL METHANOL BATCH + FED BATCH FED-BATCH 29 Feb-19 XS™ Pichia G1-3 System Availability
System available as a Lonza service offering or under a Research Evaluation Agreement
. REA includes . Proprietary expression system with host cells and vectors . Comprehensive manual covers strain development and scalable fermentation regimes . Technical Support . Proprietary technology includes granted patents and pending applications
30 Feb-19 Thank you for your attention!
Questions