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Pichia Platform 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 60 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,
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