Serum-free Media for Therapeutic Cell Manufacturing – Challenges and Innovations
•
1 like•273 views
The need for high quality materials that are animal origin-free and compatible with a limited number of downstream processing steps will increase as cell therapies achieve clinical success. Large scale manufacturing necessitates transition from planar culture to technologies such as stirred tank bioreactors where culture of suspension cells or adherent-dependent cells on microcarriers is enabled. This webinar will discuss challenges and solutions to the elimination of animal-derived components from cell culture processes, with focus on human mesenchymal stromal/stem cells (hMSCs). Fetal bovine serum in particular is associated with regulatory, supply, and consistency challenges, yet a wide range of performance has been observed between different serum-free media formulations for expansion of hMSCs in planar formats. Moreover, a positive performance in static culture is not necessarily predictive of that under agitated conditions with microcarriers, highlighting ongoing challenges to the generation of a fully chemically-defined and scalable cell culture medium. Through use of pharma-grade basal media manufactured with advanced milling technology and EMPROVE® raw materials, as well as transition to serum-free supplementation and process development activities, the robust expansion of hMSCs across platforms has been achieved. Presented by Aletta Schnitzler, Senior Scientist on 5/5/16
Recommended
Recommended
More Related Content
What's hot
What's hot (19)
Similar to Serum-free Media for Therapeutic Cell Manufacturing – Challenges and Innovations
Similar to Serum-free Media for Therapeutic Cell Manufacturing – Challenges and Innovations (20)
More from MilliporeSigma
More from MilliporeSigma (20)
Recently uploaded
Recently uploaded (20)
Serum-free Media for Therapeutic Cell Manufacturing – Challenges and Innovations
- 1. Aletta C. Schnitzler, Ph.D. May 5, 2016 Challenges and Innovations ONLY FOR U.S. AND CANADA AUDIENCE
- 2. 2 1. Clinical and commercial considerations for cell therapies 2. Cell culture media manufacturing 3. Media supplements for MSCs 4. Serum-free media Performance in planar systems Performance in microcarrier systems 5. Large-scale bioreactor culture Topics
- 3. 3 Cell Therapy Is an Active Area for Clinical Trials Source: Alliance for Regenerative Medicine 2015 Annual Data Report
- 4. 4 Cell Therapy Bioprocessing Systems and Tools Customer Collaborations Media and Reagents Preferred partner for cell therapy process development services. A specific set of operating parameters must be generated for each cell, microcarrier and medium combination. Extensive process know-how in scalable volume controlled systems, for consistent, predictable cell expansion, and maintenance of cell phenotype and function. High quality raw materials meeting GMP standards that support cell therapy manufacturing. A comprehensive regulatory information package for each formulation.
- 5. 5 Innovation is required to achieve commercial success Cell Therapy Manufacturing Considerations Clinical Success Commercial successes Scalable, GMP, Closed, Economically Viable Processes Pharma grade OR GMP raw materials (media, supplements, buffers, enzymes) Innovation TODAY Collect & isolate Culture & expand Manipulate Harvest & concentrate Formulate and fill
- 6. 6 Autologous Therapies: One to One AUTOLOGOUS = SCALE-OUT Trends and details Autologous manufacturing • Customized to patient • 1 patient = 1 batch Key Needs • Automated closed manufacturing • Pharma grade or cGMP raw materials • Faster & cost-effective release testing
- 7. 7 Allogeneic Therapies: One to Many ALLOGENEIC = SCALE-UP Trends and details Allogeneic manufacturing • Off the shelf therapy • 1 dose MSCs ~100 x106 cells Key Needs • Scalable expansion systems up to 200 - 1000L • Downstream processing • Chemically defined media / serum replacement
- 8. 8 Regulatory Challenges – Materials and Reagents
- 9. 9 Cell Culture Medium The Combination of raw materials and the production process is mandatory for excellent dry powder media performance Raw Material Quality Key for optimal media performance in cell culture applications: • Formulation • Raw material selection • Documentation Production Process Directly linked with lot-to-lot consistency and cellular performance: • Milling and Mixing process • Homogeneity • Particle size distribution
- 10. 10 Advanced Manufacturing - Milling High Quality Basal Media • Fully scalable non-animal origin production line • Controlled particle size and production temperature • Full GMP (several FDA and EMEA Audits) • Inert cold milling process (liquid Nitrogen) Mill (Ø mm) 40 100 400 Batch size (kg) 0.01-1 0.5-10 100-200
- 11. 11 Advanced Manufacturing – High particle size uniformity High Quality Basal Media A B C A: Our media is a homogeneous powder B/C: Commercial CDM powder showing crystals and non-homogeneous blending Lab scale Mid scale Large Scale batch 1 Large Scale batch 2
- 12. 12 Advanced Manufacturing - Compaction High Quality Basal Media 0 0 0 0 0 0 0 0 0 0 0 0 0 000 00 Compaction •Is a granulation technology •No water is added to the process •Works with compression force only •Fixes homogeneity in place •Process Parameters: Design of compression unit Applied force Media formulation
- 13. 13 Advanced Manufacturing - Compaction High Quality Basal Media 3x Elevated Dissolution Rate Low Dust Formation Flowability Process efficiencies – Time saving, cost saving, safety and handling improvement Reduced dissolution time Reduced contamination risk Reduced transport and handling cost 60 sec (Endpoint 22 min) Powder Medium 16 sec (Endpoint 7 min) Compacted Medium Lower Bulk Volume
- 14. 14 EMPROVE® High Quality Basal Media 98% αMEM Raw Material Grades % by weight Emprove Compendial For CCM 99% DMEM Raw Material Grades % by weight Emprove Compendial For CCM
- 15. 15 Expansion of Mesenchymal Stem Cells • SAFC fetal bovine serum, GMP quality level • PLTMax® human platelet lysate, currently research grade. GMP coming soon. Passage FBS C1 SAFC Lot 1 SACF Lot 2 FBS hPL Supplementation is required for non-immortalized cells
- 16. 16 Performance across platforms Expansion of Mesenchymal Stem Cells
- 17. 17 Platelet lysate supports large-scale process Expansion of Mesenchymal Stem Cells • MSCs retain function after expansion at large scale in αMEM + platelet lysate Differentiation Identity Function Analytics
- 18. 18 Variable performance of commercialized media in planar systems Technical Challenges with Serum-free Media XF1 SF1 SF2 SF3XF5XF2 XF3 XF4 LS1 LS2 DMEM 10% FBS • Many serum-free and xeno-free media for MSC expansion are coming to market; however, highly variable performance in both planar and microcarrier systems is observed.
- 19. 19 Technical Challenges with Serum-free Media • Cell surface marker expression is generally well-supported. SF1 SF2 XF1 XF5 XF2 XF3 XF4 LS1 DMEM 10% FBS
- 20. 20 Differentiation potential is maintained Serum-free Media can Support Key Features P8 bmMSC expanded in XF1 medium prior to differentiation • Multi-lineage differentiation potential is maintained by serum-free formulations. • XF1 medium also had best growth and cell surface marker expression. Osteogenesis Adipogenesis Chondrogenesis XF1 XF5XF2 XF3 XF4
- 21. 21 Challenges and considerations Microcarrier-based Suspension Culture • Microcarrier-based culture is highly complex. • Higher hydrodynamic forces than planar culture. • Media formulation interacts with other parameters: • Sparging • Oxygen demands • Microcarrier characteristics Schnitzler et al. Biochem Eng J 108 (2016) 3-13. Solid -Liquid Mixing, North American Mixing Forum Figure 5. States of solid suspension. A) On bottom, B) Off-bottom, C) Uniform suspension
- 22. 22 Performance in planar culture is not predictive of suspension culture Technical Challenges with Serum-free Media • XF1 medium performed best in planar culture. LS1 SF2 SF1XF2 XF1αMEM PL DMEM FBS DMEM FBS αMEM PL Xeno-free and/or defined Animal-origin or undefined LS1 SF2 SF1 XF2 XF1 • Good performance in planar culture, does not predict success in microcarrier-based systems. • Xeno-free / defined formulations generally perform poorly in microcarrier-based systems.
- 23. 23 Technical Challenges with Serum-free Media 10% FBS15% XFSup1 p6 p8 • FBS and platelet lysate typically support 10-14 cumulative population doublings in 4 passages in planar format. • XFSup1 does not fully support MSC growth in αMEM alone.
- 24. 24 From planar to suspension systems Technical Challenges with Serum-free Media • XFSup1 rescues performance of XF1, a defined medium, in microcarrier-based suspension culture. Planar Microcarrier XF1 + 15% XFSup1 αMEM+10% FBS XF1 alone αMEM FBS XF1 XFSup1: 0% 5% 10% 15%
- 25. 25 Summary Process Understanding Cell Function Quality Materials • Emprove® • Advanced manufacturing • Non-animal origin • Optimize control parameters • Hydrodynamics • Role of media components • Cell identity • Differentiation potential • Immune modulation Linking function to clinical efficacy! How do we get from here… … to here? Best Practices in Cell Therapy Manufacturing, J. Carmen. (2013) The Cell Culture Dish
- 26. Senior Scientist Cell Therapy Bioprocessing MilliporeSigma Bedford, MA, USA aletta.schnitzler@emdmillipore.com