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IBC’s Recombinant Protein & Complex Biologic Development and Purification, March 5, 2010


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IBC’s Recombinant Protein & Complex Biologic Development and Purification, March 5, 2010

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IBC’s Recombinant Protein & Complex Biologic Development and Purification, March 5, 2010

  1. 1. Virulence Factor-Based Drug DiscoveryA Novel Approach for Identifying Drug Targets for Autoimmune and Inflammatory Diseases David Bienvenue, Ph.D. VLST Corporation
  2. 2. Viral Logic Systems Technology VLST Office view: Office view: July 8th, 2009, 3:43 PM The other 364 days of 2009• Privately-held company founded in 2004• ~35 Employees• Focused on exploiting viral evolution to develop novel biotherapeutics• Based in Seattle, WA
  3. 3. Virulence Factors as a Novel Route to Therapeutics• Some viral proteins modulate/suppress host immune system• Facilitate viral infection and influence severity of disease• Can be homologous or unrelated to host genes• Targets of viral proteins validated as treatment methods for autoimmune/inflammatory illness Drug Development Strategy Identify Identify Define biologic Develop therapeuticsvirulence cellular consequences mimicking factors targets of interaction virulence factors
  4. 4. Discovery of Soluble Viral TNF Receptor Key Step in Development of Enbrel® TNFR2 Enbrel® (p75) Shope fibroma (Entanercept) virus-T2 38 % sequence identity FcSmith et al (1990) Science 248: 1019, Smith et al (1991) BBRC 176: 335
  5. 5. Cytokines, Chemokines and Their Receptors Encoded by Herpes VirusesAlcami (2003) Nat RevImmunol 3: 36
  6. 6. Genomic Scale Search for Viral Virulence Factors Analyzed >200 viral genomes •Pox >18,000 viral proteins •Herpes in VLST database •Adeno •Asfar Bioinformatic Expert System>6,000 viral protein clusters of similar Selection Criteria proteins •Topology – anchor, secreted, transmembrane •Homology to human proteins •Species infected by virus •Pfam motifs >600 putative virulence factors •Non-essential for viral replication identified and queued for screening
  7. 7. Identification of Host Targets of Virulence FactorsBioinformatic mining for Transiently express affinity- Target virulence factors tagged viral proteins identification by LC-LTQ MS Bind target(s) from supe and lysates from VF immune-related cell lines, using tandem VF affinity tag Synthesize viral genes VF
  8. 8. Viral Protein ExpressionTarget Discovery is a Numbers GameProteins expressedProteins screenedTargets identified• Maximize number of viral factors going into screen• Minimize effort, reagents spent on non-expressors• Evaluate different vectors and cell lines with Freestyle transfection reagent• Scale up via technology, not FTE’s!
  9. 9. Effect of Cell line, Vector in Freestyle Comparison of Day 4 Titers Cells: 60 56.3 51.9 50.5 293-EBNA 50 40.9 43.1 43.6 40.6 40.0 45.2 293-F 40 36.8 CHO-Fug/mL 30.7 30 26.7 20 CHO-EBNA 10 0 Vectors F- GS F GS 9 S F 409 EF In-house - 409 - GS 09 HEF F- 40 CHE - CHE EB- G EB- 4 F- CH -EB -EB- 293F 293F EB- C GS CHO CHO -EB 293F 293- CHO 293- CHO CHO CHEF 293- CHO •Same gene in different vectors and cell lines •Similar expression levels with all combinations tried
  10. 10. N- And C-Tagged Virulence Factors To Increase # Identified Targets Affinity Resin HAC Tag• “HAC”- tandem affinity tag on one end of virulence factor• Position of HAC may block binding, affect expression• Gene synthesize both N- and C-term. tagged vectors• >90% express at least one version, ~20% increase over expressing C-tag alone• Increases probability of identifying targets, in some cases, only one version binds target
  11. 11. 24-well Shaker Plate Prescreen • Reagent, time, effort wasted on large- scale transfections if viral protein doesn’t express • Developed expression pre-screen ofCHO-EBNA transients Western blot expression using 24-well shaker plates • Can be used to make relative comparisons between different expression constructs, media, etc.
  12. 12. Platform Highlights•Identified numerous immunologically relevanttargets •Validated targets of 4 approved, 11 investigational drugs•Partnership with Novo Nordisk in 2008 to provideresearch targets•Therapeutic programs based on viral targetsentering the clinic in 2010/2011
  13. 13. Therapeutic Proteins Based on Virulence Factor PlatformPossible approaches:•Recombinant viral proteins to treat autoimmune diseases •Focus on acute indications to avoid immunogenicity from long-term dosing •Not currently being pursued•If virus makes a homologue to a human protein (eg. solubleviral TNFαR), use recombinant human protein•Develop antibodies to bind to/block the targets of viral proteins
  14. 14. Challenges of Non-mAb Therapeutics• mAb-like expression levels may not be achievable• mAb-like purification process steps may not be feasible• Platform processes may not be available• More complex glycosylation patterns• Greater stability issues with non-native structures/fusion proteins• Despite potential challenges, unique therapeutic approaches makes the effort worthwhile
  15. 15. Case study: VLST-007 Human Viral•Viral protein interrupts signaling by formingheterodimer with human transmembrane proteinVLST’s approach•Make soluble Fc-fusion protein of bind to and blocksignaling through binding partner
  16. 16. Case study: VLST-007• Extracellular domain fused to IgG1 Fc• 7 putative N-linked glycosylation sites (14 in dimer) – Process may impact glycoforms, potency• Use “mAb-like” process, but customize as necessary – Anion exchange flow through step not possible• Initial process development performed at VLST, tech transfer of process and reagents to CMO• Collaborative effort to trouble shoot, assess impact of changes on product quality/activity
  17. 17. VLST-007 Development Challenges CEX Ligand-Dependent Aggregation Fractogel FractogelMabselect SP XL Eluate SO3(S) Eluate SO3 (M) Eluate Eluate •Strong CEX ligands induced product aggregation, as observed on SEC-HPLC •Recommended that CMO switch resin (after tech transfer had already begun) •CMO accommodated change with no impact to time-line GE CM FF TOYO CM Eluate 650M
  18. 18. VLST and CMO Develop mAb-Like, Scaleable cGMP ProcessCHO Fed-batch Depth filter Low pH viral Nanofiltration>1 g/L inactivation clarification ProA C H+ H+ H+ H+H+ E X HIC BDS TFF •10L PD process yield: 75% •400L Engineering run process yield: 78%Confidential
  19. 19. Leveraging Technology to Boost Titer CHO XD® Comparison 2L, 50L XD® and Fed-Batch 10 •DSM performed evaluation, utilized 9 same media and feeds as VLST’s GMP process 8 •No extensive custom mediaProduct concentration (g/L) 7 optimization necessary 6 •Titer Boost: From 1.2 to 9.2 g/L 5 in 12 days (7x) 4 50L XD 3 2L XD Fed-batch 2 1 0 0 2 4 6 8 10 12 14 16 18 Time (days)
  20. 20. Case Study: VLST-018• Mechanism validated by viral biology- multiple viral homologues mimic a membrane-bound human protein• Generate soluble Fc-fusion of extracellular domain• Multiple N-linked glycosylation sites• Generate production cell lines, perform PD and tech transfer to CMO
  21. 21. Generation of Production CHO Cell Lines •Codon-optimize gene, and clone into two different proprietary expression vectors •Screen transfectants via Clonepix •Use to weed out low-expressing clones •Permitted thousands of colonies to be pre-screened •Titer results significantly better (40%) with top GS clones, will utilize for future cell line campaigns
  22. 22. Purification Development High-throughput Screening96-well plate Chromatography •Screen multiple conditions quickly and easily •Facilitates use of DOE •Rapidly identified lead CEX resin based on capacity, using little protein
  23. 23. Lessons Learned/Conclusions•Leverage new technology, rather than addingFTE’s, to do PD better, faster •24-well prescreen •Alternatively-tagged constructs •Clonepix •Alternative cell culture methods, XD, perfusion to increase titer •Utilize vendor’s expertise and help if possible •Plate-based chromatography development
  24. 24. AcknowledgementsProtein Sciences Group• Jeff Bartron• Chris Tompkins• Laura Hajny• Patrick Mosher• Ryan Kelly• Ryan MerrillBioinformatics and Proteomics• Stefan Ponko, Ph.D.• Ajamete Kaykas, Ph.D.