BIOLOGICS DEVELOPMENT From Discovery to Manufacturing

Transcription

1 BIOLOGICS DEVELOPMENT From Discovery to Manufacturing

2 About LakePharma LakePharma is the leading US-based biologics company specializing in antibody and protein, engineering, cell line development and protein production. We enable the discovery and development of the biologics of tomorrow. Discovery Engineering Development Manufacturing LakePharma started with two people in 2009 with the purpose of making drug research, development and production more nimble, transparent, and efficient. Today, LakePharma is among the fastest growing companies in the world, and a leader in providing integrated services and expertise for biotechnology and pharmaceutical companies. With a new MicroBiomanufacturing Center joining the Antibody, Development, and Vector Centers, we can now offer end-to-end services including integrated contract development and manufacturing capabilities in vital US biotech clusters. - Hua Tu, Ph.D., Founder and CEO 2

3 Fully Integrated Technology and Services, from Lead Discovery to Phase I Antibody discovery, Antibody engineering, Antibody production Process development, Product development, Reagent manufacturing Molecular biology, Cell culture, Protein science DS manufacturing, DP manufacturing, Assays and analytics LakePharma Antibody Center (CA) LakePharma Development Center (CA) LakePharma Vector Center (MA) LakePharma MicroBiomanufacturing Center (MA) Four independent technology centers All services performed in the US Dedicated client service and project management teams 24/7 secure access to cloud-based client portal built on the RENKOM Enterprise Software Platform 100% service business model, no internal drug development programs Inc fastest growing private companies in 2015 and 2016 Technology networks and partnerships: - LakePharma Antibody Technology Network (LATN) - LakePharma Microbial Fermentation Network (LMFN) - LakePharma CMC Expert Network (LCEN) - LakePharma Bioanalytical Network (LBAN) 3

4 Therapeutic Grade Monoclonal Antibody Discovery in vivo Fully customizable antibody discovery campaigns Immunizations - UberCell immunizations for functional mabs against membrane targets - HT-Hock immunization - Protein and peptide based RIMMS7 immunization - DNA-based immunization - Tolerance breaking immunization Access to Trianni and other transgenic mouse platforms Antibody Recovery - Screen for affinity, cross-reactivity, function - Novel B-cell enrichment protocol for increased hit rates - Electrofusion for high fusion efficiency - High-throughput 384-well screening Hybridoma sequencing, antibody reformatting, recombinant antibody expression and purification Delivery of clonal, functional mabs with V-region sequences, hybridomas, and functional data RIMMS7 Rapid Protein Immunization For High Affinity mab Discovery Titer against human target protein 384-well plate ELISA screening of hybridoma supernatants to identify species cross-reactive antibodies Titer against cyno target protein 4

5 Antibody Development by Phage Libraries Species cross reactivity Liability assessment Bispecific antibodies Epitope binning and mapping De novo antibody discovery LakePharma Antibody Engineering Kinetic assessment First in class phage libraries (XOMA) Antibody reformatting, isotype and subtype switching Affinity maturation Custom library construction and screening Xoma Fab Phage Library Picomolar affinity antibodies routinely selected Very large (> ), fully human, high percentage of open reading frames Leads from this library in multiple clinical programs Three tags (V5, 6His, c-myc) Naïve Fab Phage Libraries > 10 9 diversity 30 fully human HC frameworks, 20 LC frameworks kappa lambda even distrubution Yeast Display Libraries Antigen Specific Immune Libraries Llama, mouse, rat, rabbit, and chicken libraries Custom library construction and screening: antibody, protein, and peptide 5

6 Antibody Engineering Services Antibody Humanization Humanization and germlining Sequence liability analysis and verification Affinity Maturation Light chain shuffling (Fab or scfv libraries) Heavy or Light chain CDR mutagenesis Bispecific Antibodies Several scaffolds with freedom to operate Access to proprietary scaffolds via LakePharma licensing High-throughput Design and Screening HighRes Biosolutions automation Carterra LSA for antibody characterization 6

7 mau Transient Production in HEK293 and CHO Cells Molecular construction into LakePharma s proprietary high-expression plasmids or custom construction into client plasmids IgG, bispecific antibody, Fab, scfv, IgM, and more LakePharma-optimized HEK293 and CHO production - Tuna293 TM : mg/l, typical antibody expression - TunaCHO TM : mg/l, typical antibody expression Additional platforms: Expi293 TM and ExpiCHO Flexible production volumes from 30 ml to 20 L culture Affinity and custom antibody purification Elution Profile CE-SDS Elution Wash Loading 7

8 96 Batch High-throughput Antibody Production Recombinant antibody production (HEK293) Average yield = 0.7 mg from 10 ml High-throughput gene synthesis, cloning, expression, and purification LakePharma HEK293 and CHO production processes - Tuna293 TM : average antibody yield 0.7 mg from 10 ml production - TunaCHO TM : average antibody yield 4.2 mg from 10 ml production Fast turn around time in 5-7 weeks from antibody sequences to purified samples Ideal for rapid antibody expression and screening Also available for Fc-fusion proteins, bispecific antibodies, etc. Intact Mass by Mass Spec QC included for standard IgG productions Recombinant antibody production (CHO) Average yield = 4.2 mg from 10 ml A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 C2 D2 E2 F2 G2 H2 A3 B3 C3 D3 E3 F3 G3 H3 A4 B4 C4 D4 E4 F4 G4 H4 A5 B5 C5 D5 E5 F5 G5 H5 A6 B6 C6 D6 E6 F6 G6 H6 A7 B7 C7 D7 E7 F7 G7 H7 A8 B8 C8 D8 E8 F8 G8 H8 A9 B9 C9 D9 E9 F9 G9 H9 A10 B10 C10 D10 E10 F10 G10 H10 A11 B11 C11 D11 E11 8

9 CHO Stable Cell Line and RCB Development LakePharma proprietary technology, full freedom to operate. GS Stable Vector Stable Pool Generation Stable Clone Generation Limited Dilution + Titer Screening Stable Subclone Generation Limited Dilution + Titer Screening RCB RCB and Product Characterization MCB Robust performance CHO-K1 GS knockout line enables stronger GS selection - Pool can reach > 1 g/l in shake flasks - Simplified process for high titer clone selection (> 4.5 g/l) - Imaging by Solentim to confirm monoclonality Start to finish of the RCB in 6 months Compatible with many chemically defined commercial media High tolerance with shear (foam and tip speed) Generational stability generations 9

10 Antibody and Protein Characterization LakePharma offers comprehensive analytical characterization, from sequence to structure to function. Developability Assessment Stability Study Formulation Study Discovery Engineering Development Antibody de novo sequencing Affinity screening (off rate) by Octet Epitope binning Thermostability screening Polyspecificity screening Cell-based functional screening Intact mass analysis Sequence liability analysis Affinity characterization and FcγR and FcRn binding profile Epitope binning Cell-based functional characterization Peptide mapping by Mass Spec PTM and glycan profile by Mass Spec Custom quantification for single cell cloning Buffer formulation and stability testing Aggregation propensity Cell-based functional characterization 10

11 Immune Checkpoint Assays Antibody Functional Characterization Affinity measurement - BLI, SPR, FACS, ELISA FcγR binding assays and FcRn binding assays Epitope binning and mapping Effector function assays - ADCC, ADCP, CDC Primary T-cell functional assays - Stimulation and proliferation assay - Mixed lymphocyte reaction - T-reg suppression assay - CMV Recall Immune checkpoint assays - CTLA-4, PD1-PDL1, GITR, OX40, etc. Custom cell-based, reporter-gene, or receptor-ligand assays 11

12 BioAnalytical Characterization Analytical Characterization Affinity assays including Octet, SPR, ELISA, and FACS Purity assays including CE-SDS and SE-HPLC Aggregation assays including SE-HPLC and DLS Thermostability assays including DSF and DSC Charge variant profiling by ice3 Mass spectrometry analysis including glyco-profiling, PTM analysis, intact mass Antibody de novo sequencing by mass spectrometry Developability Assessment Package In silico sequence liability assessment followed by mass spec verification Integrity and stability check using stress tests PK readiness analysis (polyspecificity assay) Light Chain Heavy Chain 12

13 Comprehensive Molecular Biology Services Gene synthesis all constructs fully sequence verified Vector construction and molecular cloning - Ligation, PCR, site mutagenesis, gateway, and other custom cloning Endotoxin-free plasmid DNA production - Midi, Maxi, Mega, Giga, up to 1 gram quantity - High-throughput 96-well miniprep - 23-hour DNA purification mrna/in vitro transcription: up to 10 mg/run Gene expression and copy number analysis Viral Vector Platforms: Lentivirus and Adeno- Associated Virus 13

14 Gene-to-Protein Services Mammalian Expression Transient production in suspension HEK293 and CHO cell lines Stable cell development and production Flexible production volumes (up to 175 L in 5 weeks) Hybridoma expression up to 175 L Adherent cell scale-up up Viral Vector Platforms: lentivirus and Adeno- Associated Virus Insect Expression Sf21, Sf9, High 5, and T.ni cells 30 ml P0 stock with titers > 10 8 pfu/ml Expression scouting to optimize quality and yield Variable scales (10 ml to 175 L) with industry leading turnaround times Experienced team who pioneered key advancements such as: - Titer-less expression with Infection Kinetics Monitoring (IKM) which saves weeks in expression testing - Titer-less Infected Cell Preservation and Scale-up (TIPS), an affordable and superior way to preserve and scale up baculovirus Enriched baculovirus particles for polyspecificity screening Affinity and custom purification, covering 0.01 L to 175 L expression volume 14

15 Gene-to-Protein Services E. coli Expression Gene synthesis, molecular construction, and cloning into custom or in-house expression vectors E. coli strain creation and expression optimization Scale up services up to 100 L in flask or 500 L in fermentor Industry leading turnaround time: up to 50 L in 2 weeks Yeast Expression Pichia pastoris or Saccharomyces cerevisiae Strain creation and expression optimization Scale up services up to 100 L in flask or fermentor Industry leading turnaround time: up to 20 L in 5 weeks Custom scalable purification: multi-step enrichments from L to 40 L column volumes, supporting up to 500 L expressions. 15

16 Protein Chemistry Services LakePharma offers custom conjugation and labeling services for up to gram levels of proteins. Custom Conjugation Services: Oligonucleotide-protein conjugation Protein-protein conjugation - HRP conjugation - PE conjugation - APC conjugation Peptide-protein conjugation Protein conjugation to resins Custom Labeling Services: Fluorophore labeling Biotinylation - In vitro - Site specific PEGylation Custom Proteolysis Services: Antibody fragmentation - Fab generation - Fc generation - F(ab) 2 generation 16

17 Customized Assay Design and Development Services Cell-Based Assays HUVEC/primary cell/cell line workflows Customizable cytotoxicity 96/384-well Cytokine stimulation and release with multiplex detection methods Metabolic labeling pulse/chase workflows with LC/MS and radiometric methods Reporter cell line development and screening Flow cytometry/facs/confocal microscopy Assay Formats/Readouts Custom ELISA development (TMB/MSD ECL) Lum/fluor formats (luminescence, FL, FP, FRET, TR-FRET, HTRF, UV-Vis Abs.) Binding kinetics analysis, Ki determinations SPA and radioligand binding (cell-based, cell-free) LC/MS-based analysis (high throughput and custom method development) qpcr: viral quantitation, gene expression, splice variant analysis, multi-analyte workflows 17

18 Process Development and Biomanufacturing L SUB from Sartorius Upstream process optimization Downstream process optimization 50 L to 500 L single-use bioreactor production Production of drug substance for in vivo animal studies Production of GMP drug substance for Phase I clinical trial Development of a high-performance process suitable for clinical supply Process transfer to CMO Ambr15 mini-bioreactor from Sartorius 2L SUB from Sartorius 18

19 Formulation Development and Stability Study Pre-formulation and formulation support upstream and downstream process development High-throughput screening of buffers and excipients utilizing DSC and DLS Design of Experiment (DoE) to identify optimal formulation and important variables Stress testing including heat incubation, freeze/thaw, and oxidative stress Generation of stability data to support shelf-life of CTM Accelerated and long-term stability studies for DS and DP Characterization of reference materials 19

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