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SAFC Biosciences Raw Materials Characterization Initiative PowerPoint Presentation
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SAFC Biosciences Raw Materials Characterization Initiative

SAFC Biosciences Raw Materials Characterization Initiative

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SAFC Biosciences Raw Materials Characterization Initiative

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  1. SAFC BiosciencesRaw Materials CharacterizationInitiative Michael Wathen

  2. Why Raw Materials? • Raw Materials Characterization was identified as the highest priority by our customers • Produce a quality product with minimal variability • Understand impact of raw material changes • SAFCB has major raw material initiatives in several areas • Raw Materials Characterization Initiative in R&D • Coordinating with Supply Chain and Quality initiatives • Key values for our Customers • Reduced variability in media performance • Improved response to technical inquiries • Improved ability to resolve investigations • Reduced customer resources spent on raw materials

  3. Raw Materials Characterization InitiativeOverview of Primary Objectives Raw Material Initiative Raw Material Characterization Raw Material Effects in Complex Media Components (AI) Physical Biological Impurities Complex Simple Objective Determine effects resulting from physical changes in components Database predicting chemical reactions with mixtures of components Database predicting effect of RM changes on media performance and metabolic pathways New specifications to minimize risk of impurity effects Analytical markers predicting erformance Outcome Reduce variability in product performance Special testing to allow higher grade RM offering Rules on permissible modifications. More relevant specifications for solubility Improved stability. Improved concentrate feasibility Improved media development

  4. Raw Materials Characterization Projects • Impurities • Analytical and biological evaluation • Evaluate variability in lots and vendors • Biomarkers for Complex Components • Starting with soy hydrolysate • Analytical characterization of lots with range of performance • Test analytical markers for predictive value • Characterization of Physical Properties • Variability in crystal structure—relationship to solubility • Effects of salt or hydration changes on biological activity

  5. Raw Materials Characterization InitiativeOverview of Support Projects Raw Material Initiative Searchable Database Assays Prioritizing Raw Materials Projects Analytical Biological Objective Common database with search capabilities to link assay data with formulations or components Prioritized list of raw materials for evaluation by project teams Sensitive assays to identify RM’s, contaminants and changes in product quality Sensitive assays to detect changes in cell growth, productivity, or product quality Outcome Ability to detect changes in RM’s and the effect of those changes on the cell and its recombinant product Ability to identify trends in data based on modifications in formulations Project teams will focus on highest risk raw materials

  6. Raw Material Characterization InitiativeDeveloping a Prioritized List of Raw Materials • Goal • Ensure project teams focus on the highest risk raw materials • Approach • Obtain a master list of raw materials used by SAFCB • 331 different raw materials in SAFCB inventory • Develop ranking system for rating potential risks associated with raw materials • Rank raw materials and develop a “Top 50” list

  7. Prioritized List of Raw MaterialsRisk Factors and Weightings

  8. Prioritized List of Raw MaterialsTop 20

  9. Raw Materials Characterization InitiativeAssay Development • Analytical assays • Raw material identification assays • Impurity profiling and identification • Biological assays —Sensitive cell assays to detect differences in: • Growth • Productivity • Product quality

  10. Analytical Assay DevelopmentReverse Phase HPLC Impurities

  11. Raw Materials Characterization InitiativeImpurity Profiling and Identification • What are the major impurities in each RM? • What is the lot-to-lot variability in these impurities? • Does the impurity profile change with different vendors? • What is the “no-effect” range for the major impurities relative to growth, productivity and product quality? • Will the impurities effect biological activity within the range of concentrations observed in the raw material

  12. Raw Materials Characterization InitiativeConsiderations for Biological Assays • Cell lines • Multiple cell lines relevant to biopharmaceutical industry • Robust with low variability • Sensitive to differences in raw materials • Media • Chemically defined preferred • Rich vs. lean? • Assay systems • Balance high throughput and low variability • Assay parameters • What is the most sensitive parameter for growth? • What are the useful product quality parameters?

  13. Assay System Cell lines (3) CHO K1 CHO GS EB66 CHO DG44 Per C6 Media: Rich vs. lean medium CHO K1: CD fusion CHO GS: CD fusion EB66: GRO-I DG44: 1+8 lean Per C6: Ex-Cell VPRO 14561C Counting Method Cedex Guava 96-well Culture Vessel TPP tubes 24 deep well plates Raw Materials Characterization InitiativeBiological Assay Qualification

  14. Raw Materials Characterization Initiative Biological Assay Qualification Qualification Parameters • Assay Parameters: Seed density, assay time, passage number, adapted vs. non-adapted • Reproducibility: Operators, equipment (Cedex, shakers) Challenge Studies • Positive Effectors • Lysine, calcium pantothenate, folic acid, insulin • Negative effectors • Triton X-100, sodium selenite, trace element (Copper) • Neutral (osmo effect) • NaCl • Combination • 1 positive + 1 negative effector

  15. Raw Materials Characterization Initiative Biological Assay Qualification • Cytotoxicity assay model • Dose response model: IC50/EC50 • Characterize raw materials by concentration response, inflection point, slope • Growth Phase IVCD (Days 1-7) • Assay Criteria • Reproducible and sensitive quantification • Mid-High cell yields in batch culture (>3E6/mL) • High % viability (>95 %) • DT <30 hours • Low cell aggregation throughout growth & stationary phase (<15 % CV)

  16. Biological Assay Qualification Detection of Positive and Negative Effectors • RMAQ002 • Purpose • Evaluate DG44, CHOK1, and DUX CHO cell lines in media formulation containing different lots of Hydrolysate Media • Hydrolysate Lots at both 5g/L and 2g/L • 8E0258 “Good lot” • 6L0359 “Bad lot” containing known impurity • System • TPP tubes at 30mL volume

  17. Biological Assays IVCD Values for Good vs Bad Hydrolysate

  18. Biological Assay Qualification Detection of Positive and Negative Effectors • Conclusions • CHOK1 • Robust cultures with little variability throughout growth curve. • Results showed a good response to both the good and bad lots of Soy Hydrolysate. • DG44 • Cultures showed more variability throughout growth curve. • Results showed a good response to the presence of hydrolysate, but did not show inhibition with the bad lot. • DUX • Aggregation occurs early in culture • At times cultures didn’t grow well enough to make it through adaptation

  19. Biological Assay QualificationDetermining EC50 of Positive Effector (Lysine) VCD of CHO K1 Cells in Media Containing Different Conc. of Lysine

  20. Biological Assay QualificationDetermining EC50 of Positive Effector (Lysine)

  21. Biological Assay QualificationDetermining EC50 of Positive Effector (Lysine) EC50 = 46 mg/L Rsqr = 0.97 Hillslope = 0.58

  22. Example of Raw Material CharacterizationTyrosine Investigation • Customer complaint of poor media performance linked to specific vendor (Vendor 1) of tyrosine by analysis of batch records. Media using alternative vendor (Vendor 2) performed to expectations • No differences observed by RP-HPLC, SEC or UPLC-MS • Review of manufacturing process indicated difference between vendors in final drying method • Spray drying vs. tray drying • Multiple lots of L-Tyrosine from 3 different vendors were evaluated for differences in biological effects

  23. Tyrosine InvestigationVCD using different sources of tyrosine

  24. Tyrosine InvestigationProductivity using different sources of tyrosine

  25. Amino acid analysis indicated reduced levels of L-Tyrosine 2Na 2H2O for media containing Vendor 1 material following filtration Direct correlation between amino acid concentration remaining after filtration of media and productivity in cell culture assay Tyrosine InvestigationAmino Acid analysis of media

  26. Tyrosine InvestigationAnalytical Characterization of Crystal Structure • Observed differences in solubility of L-Tyrosine from different vendors led to evaluation of physical properties • Utilized Pharmorphix which is a division of Sigma-Aldrich specializing in pharmaceutic properties • Evaluated 3 lots each from Vendors 1 and 2 • X-ray particle diffraction (XRPD) for crystal structure • Differential scanning chromatography (DSC) for melting point of crystals • Ion chromatography for quantitation of Na+ ions

  27. Vendor 1 Vendor 2 Tyrosine Investigation X-Ray Particle Diffraction • Distinct XRPD patterns indicate different crystal forms between Vendor 1 and Vendor 2 • XRPD pattern consistent between lots for Vendor 2 • Variability in crystal forms observed with different lots from Vendor 1 • Multiple crystal forms observed in one lot from Vendor 1

  28. Tyrosine InvestigationConclusions • There was a negative biological effect in both growth and productivity for CHO GS cells grown in media containing the L-Tyrosine from Vendor 1 compared to other vendors • Poor solubility caused reduced L-tyrosine from Vendor 1 in filtered media • Crystal structures of powder are different for Vendor 1 and Vendor 2 L-tyrosine • Differences observed using both XRPD and DSC • Probably due to the different final drying methods used by the manufacturers • Difference in crystal structures probably linked to solubility differences • Vendor 1 has been removed as an approved vendor for L-tyrosine

  29. Raw Materials Characterization ProgramTimeline Preliminary Projects Characterization Projects for Top 50 Raw Materials

  30. Raw Material Characterization ProgramSummary • This is a long-term initiative • Involves complex scientific issues • Will utilize both biologists and chemists • Goals of initiative • Industry leaders in knowledge of our products • Translate knowledge into value for our customers