Expression of Next Generation Biologics Requires Next Generation Expression Systems

Transcription

1 Expression of Next Generation Biologics Requires Next Generation Expression Systems Joachim Klein, Ph.D., Associate Director, Head of Strain Development and Cell Banking Georg Blaser, Ph.D., Associate Director, Commercial Development

2 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 forwardlooking 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

3 Overview Introduction to Next Generation Biologics XS Expression Technologies XS Pichia Case Studies GS Gene Expression System GS System Case Studies Accessing the Systems Summary 3

4 Why Next Generation?

5 Biologic Pipelines are Evolving Novel disease targets and biological mechanisms require new molecule formats There is demand for target therapeutics with improved efficacy Pharma pipelines evolving to address MAb competition, biosimilars and payer challenges Pipelines moving from standard format MAbs to bispecifics, novel glycoproteins and other complex molecules New molecular formats provide patient benefit and demonstrable clinical cost effectiveness Mol Immunol Oct;67(2 Pt A): doi: /j.molimm Epub 2015 Jan

6 Next Generation Biologics the Challenges Production challenges - complex molecular structures with poor stability and similar product / contaminants Upstream and downstream challenges platform approach is not feasible Analytical challenges need for HT screening to meet desired development timelines Expression challenges sufficient quantity at required quality No expression system is currently addressing all requirements Molecules are expressed in either Ma or Mi systems Bispecific Expression Either 25% Mammalian 47% Microbial 28% Citeline Analysis, Feb

7 XS Expression Technology

8 Next Generation Molecules Require More Expression Options 8

9 Different Hosts for Different Products 9

10 The XS Toolbox A Broad Portfolio XS Technologies E. coli Pichia Bacillus Sugar Inducible Depletion Inducible IPTG Inducible Glucose Regulated Strong Constitutive Methanol Inducible GAP Constitutive Sugar Inducible Auto Inducible 10

11 XS Pichia pastoris Systems for Expression of Next Generation Molecules

12 Tuning the Pichia Secretion Capacity Host strain Lonza proprietary and nonproprietary Pichia pastoris strains Folding and secretion Helper proteins Translocation Signal sequence Genome Integration Promoter Integration site Copy number Codon bias of the target gene Delic et al. (2013) FEMS Microbiol Rev. 37(6):

13 Novel Glucose-Regulated Promoter Simple screening and straightforward fermentation regimes High titers in short total fermentation time G1-3 GLYCEROL BATCH GLUCOSE FED-BATCH AOX1 GLYCEROL BATCH + FED BATCH METHANOL FED-BATCH Supernatant titer + 80% Total fermentation time - 125%

14 XS Pichia Novel Signal Peptides Standard MF-EAEA- Protein 1 kdasizedifference Lane Strain Signal peptide 1 Size marker 2 Reference standard 3 8 PF233 MF-EAEA 9 14 SMD1168 MF-EAEA PF233 SP Lonza SMD1168 SP Lonza Standard SP-Protein no size difference New signal peptide High productivity Alternative to alpha mating factor signal peptide increasing the probability of creating the best expression clone Drives high titers in the culture supernatant Better quality No N-terminal variants due to a precise signal peptide cleavage 14

15 Screening Workflow XS Pichia

16 Combinatorial Screening with XS Pichia Optimized work flow for rapid identification of the best combination for successful product expression Stage Variable No. of Options Gene synthesis Codon bias tbd Vector construction Promoter 5 Signal sequence > 3 Integration site 2 Transformation Host 2 Primary screening No. of clones > 11 Media composition > 2 Clones > 660 Microtiter plate cultivations >

17 Optimized Work Flow from Gene to Non-GMP Product Material Supply Gene Optimization & Synthesis 2-4 weeks GeneArt or DNA 2.0 Primary Screening 4 weeks Cloning and sequence verification 24/96 deep-wells Analytical options: SDS-PAGE, mce (Caliper), ELISA (Gyros), Western, biolayer interferometry (Octet) Secondary Screening 2 week Parallel 1 L DASGiP fermentations Top performing clones selected Analytical options: SDS-PAGE, mce (Caliper), ELISA (Gyros), Western, biolayer interferometry (Octet) Milligram quantity Supply Option 2 week Affinity chromatography Process Development 17

18 Case Studies for XS Pichia pastoris Next Generation Biologics

19 Case Study One i-bodies Target protein i-body amino acid sequence provided by AdAlta (Victoria, Australia) Single domain antibody-like molecule of human origin Aim Alternative production platform to Escherichia coli for i-bodies Screening in Microtiter Plates: 24 combinations Host strain: Wild type mut + without any helper factors Promoter: P G1, P G1-3, P AOX1, P CS1 Signal peptide: -mating factor, Lonza signal peptide Integration site: Multi-copy locus, AOX1 locus Screening: 11 clones per construct 19

20 Case Study One i-bodies G1-3 AOX1 1 L 1 L Screening titer in MTPs G1-3: 77 mg/l (83-fold increase) < AOX1: 102 mg/l (35-fold increase) High titer (> 5 g/l) and short product retention time in the supernatant Total fermentation time of 60h comparable to «bacterial» systems High viability and target protein purity with G1-3 20

21 Case Study Two Antibody Mimetic Molecules Target protein Three domain antibody mimetic molecule amino acid sequence provided by Affibody (Stockholm, Sweden) Aim Microbial secretion platform for antibody mimetic molecules that enables simplified purification Screening in MTP - 2 combinations Host strain: Wild type mut + without helper factors Promoter: P G1-3 Signal peptide: -mating factor, Lonza signal peptide 2 Integration site: Multi-copy locus Minimal screening: 11 clones per construct 21

22 Case Study Two Antibody Mimetic Molecules Screening titer in MTP for G1-3: 37 mg/l (92-fold increase) Direct implementation of novel (speed) fermentation regime without product specific adaptation Biomass < 120 g/l, total fermentation time of 48 h at 30 C 3.4 g/l of product, > 70% purity SDS PAGE Results 22

23 Case Study Three Single Domain Antibodies Target proteins Antibody fragments consisting of a single monomeric variable antibody domain Aim Novel Pichia production platform for single domain antibodies Screening in MTP (72 combinations) Host strain: Two wild type mut + strains, one mut S strain Promoter: P G1, P G1-3, P G6, P CS1, P AOX1, P GAP Signal peptide: -mating factor, Lonza signal peptide 1+ 2 Integration site: Multi-copy locus, AOX1 locus Screening: 11 clones per construct 23

24 Case Study Three Single Domain Antibodies Screening titer in MTP for AOX1: 150 mg/l (10-fold increase) Secondary screening without product specific adaptation Biomass < 70 g/l, total fermentation time of 76 h at 30 C > 1.5 g/l of product, > 70% purity 24

25 Using XS Technologies (Pichia) Is Proven to Improve: Productivity, Speed and Process Robustness Productivity Up to 10 g/l for Next Generation Biologics expressed in Pichia Methanol-free options with superior productivity to AOX1 Alternative secretion signals to improve product quality Speed Hundreds of clones are tested in a combinatorial screening to identify the best production clone within 4 weeks Optimized PD workflows and automated systems can enable delivery of clinical grade product in less than 9 months Speed fed-batch fermentation (60 hours versus 120 hours) Simple, Scalable & Robust Processes Robust platform fed-batch fermentation and recovery protocols ensure scalability Methanol-free, simple induction via carbon-source shift Soluble and secretion expression results in simpler recovery and downstream unit operations 25

26 GS Gene Expression System

27 Next Generation Biologics Challenges and Considerations Next Generation Biologics may not be a straightforward fit to a classical (often mab-based) platform process. This can be true for : Vector construction (Genetic design and configuration) Clone selection process (requirement for a HT titre screening method, number of clones required to find one with desired PQ attributes) Upstream Processing (culturing conditions, duration, additives, glycosylation, other post-translational modifications, etc.) Downstream Processing (lack of affinity chromatography, ph instabilities and pi consideration for viral inactivation and purification resins) Analytics (size, charge, hydrophobicity etc. can impact suitability of otherwise routine platform methods.) Regulatory/QC (support for novel classes, compatibility of documentation and suitability of testing) Formulation (ph stability, final concentration, application route) 27

28 Common Strategy Discovery to Development Selection: Expression host Upstream characteristics Downstream strategy Computational Screen/ Engineering Risk factor determination Guidance for purification/ formulation Few weeks Early Material Supply Use of surrogate models Enable early purification, analytical and formulation development Tox study Few weeks to months Product Quality Analysis/ Definition 28

29 GS System Experience with Next Generation Biologics Next generation biologics 29.4% Bispecific mabs mini-bodies One-armed mabs Fc-fusions Albumin-fusions Fab -fusions Other fusions mabs 57.3% Fabs 4.0% Enzymes 4.0% Hormones 5.2% The GS mammalian expression platform has wide range of applicability The data above represents >600 molecules expressed by Lonza, Cambridge for our Clients at early development stages in the last 3 years 29

30 GS System Advantages Uses material throughout discovery and development stages which is closely representative of your final product Expressed in same cell line (GS Xceed System) using same vectors Expressed in an industry proven expression system with regulatory track record Uses transient or stable pooled transfection in CHOK1SV GS-KO cells for proof of concept to ascertain expression levels of the target molecule(s) and to define CQAs as well as the identification of PTMs, production contaminants, aggregation levels, etc. 99.7% of molecules were successfully expressed in our GS mammalian expression platforms The remaining 0.3% of molecules could not be enriched to a commercially viable level due to their unusual biological functions that interfered with the expression hosts 30

31 Case Study One Complex Recombinant Protein 8 weeks Aim: Improve yield and half-life of human hormone Success criteria: Improve genetic design of expression gene to promote yield and glycosylation state Bias production to highly glycosylated isoform Isolate and purify glycosylated form of recombinant protein Approach taken: A small set of expression vectors were designed and evaluated in GS Xceed expression platform as stable pools. Bespoke 3-step purification was employed to yield highly pure glycosylated isoform. Screening of purified products identified lead genetic construct, provided sufficient amount (~0.5 g each) to determine CQAs Lead has now transferred to cell line construction as initial CQAs were met 31

32 Case Study Two Fusion Molecule 14 weeks 5 wks Aim: Assist in design and production of an Fc fusion molecule Success criteria: Maintain minimal immunogenicity profile Generate highly productive, functional fusion molecule Design scaleable purification process Approach taken: Molecule linkers were designed and potential variants assessed for potential immunogenicity Selected lead candidate was expressed in stable pooled GS Xceed system (multi-gram scale) First purification step was optimized to decrease aggregation propensities and material was supplied for successful in vitro, in vivo and toxicology studies Further material supplied for pre-formulation study and further purification development GMP production on-going 32

33 Case Study Three Modified, Bispecific mab 21 weeks Aim: Produce and evaluate platform fitness for a modified, bispecific mab Success criteria: Create highly producing expression system Design scaleable purification process Approach taken: Candidate panel expression undertaken to enable customer-selection on best format Selected lead candidate was expressed in stable pooled Xceed system (multigram scale) First purification step was optimized to decrease aggregation propensities Material was supplied for successful in vitro and in vivo studies as well as for preformulation study and further purification development GMP production completed 33

34 GS CHO Summary A significant track-record demonstrated that the mammalian GS expression system is versatile and highly suitable for high titre expression of next generation biologics (up to 0.7 g/l post purification from non-optimised stable pools in abridged fed-batch (bispecific)) Developability tools such as computational, in silico early phase assessment, selection and derisking of candidate molecules can prove instrumental to reduce attrition rates and to safeguard your molecule Screening variants/ candidates quickly by transient transfection or in microbial hosts Identify and define CQAs early to ensure product quality, safety, efficacy, and process consistency 34

35 Light Path Host Screening Mammalian CHO Cell Line GS Xceed Expression System Microbial XS Microbial Expression Systems Mammalian and Microbial Host Generation Expression Evaluation Product Assessment SDS Page Western Blot ELISA (optional) Expression Feasibility Report in 4 Weeks Many next generation therapeutics can be expressed in either mammalian or microbial systems Light Path Host Screening provides rapid identification of the best option, in terms of yield, for successful protein expression Molecules will be expressed in both our Mammalian GS Xceed and Microbial XS Expression Systems 35

36 The XS and GS System Research Evaluation Packages Patents/Access Rights Worldwide IP protection Global access by users, facilitating global product licensing Users have right to access/use Lonza s lab-scale bioreactor fed batch manufacturing process Materials Proprietary host cells Vectors GS System includes access to Media and Feeds Comprehensive manuals guiding cell line or strain development Technical Updates and Support 36

37 Lonza s Expression Technologies for Next Generation Biologics Proven expression systems for complex protein production including next generation biologics Options include REA for in-house use or development services provided by Lonza Services provide transition options from discovery development to manufacture Rapid Host Screen accelerates the selection of the optimal expression host system 37

38 Thank you for your attention! Questions / Discussion