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Kurtz biomanufacturing

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Mary Jane Kurtz presents an overview of teaching biomanufacturing to high school students.

Mary Jane Kurtz presents an overview of teaching biomanufacturing to high school students.

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  • Here is the premise of the ‘Protein is Cash’ Workshops designed to grow the local biotech/biomanufacturing network across the nation.
  • Jim to tell NVC what our vision and mission statements are: Vision : To be the nationally-recognized center of excellence that develops a world-class sustainable biomanufacturing workforce to improve the quality of life. Mission: Coordinate local and regional efforts into a national biomanufacturing education and training system to promote, create, and sustain a qualified workforce.

Transcript

  • 1. Protein is Cash:State of the Art Curriculum to Support the Bioeconomy Mary Jane Kurtz, Ph.D NBC 2 Consultant [email_address]
  • 2. Basis of the Bioeconomy
    • Central Dogma: DNA RNA Protein
    • Discovery Research (DNA Centric)
    • Process Development and Biomanufacturing (Protein Centric)
  • 3. Ten Technician Jobs Anchor Ten Biomanufacturing Departments
    • Facilities/Metrology
    • Validation
    • Environmental Health and Safety (EH&S)
    • QA
    • Upstream Processing
    • Downstream Processing
    • QC Microbiology
    • QC Biochemistry
    • Process Development
  • 4. Biomanufacturing/Biotechnology A plus for Teachers and Students
    • Integrated science education: Science Technology Engineering and Math (STEM)
    • Career pathways towards work/school focused on science with thousands of new jobs predicted in the next few years
    • Laboratory based activities = increased interest
      • More exciting ways of introducing concepts
      • Hands-on learning is more inclusive
      • State-of-the-art laboratories
  • 5. NBC 2 Products
    • The NBC 2 has worked for six years with both industry and educators to create biomanufacturing curriculum materials for multiple audiences, utilizing the latest in pedagogical technology.
  • 6. Growing the Local Biotech/Biomanufacturing Network across the Nation
    • Build the local biotech/biomanufacturing high school, community college, university and industry network:
      • Hold Protein is Cash workshop at local community college teaching biotechnology/biomanufacturing
      • Invite local biology, chemistry, physics and biotechnology high school teachers
      • Grant graduate credit through local university for workshop
      • Tour local biomanufacturer
      • Present local educators from universities and community colleges
      • Present local industry , including local supplier(s) of biotechnology and biomanufacturing equipment and supplies
    • Teach teachers the theory/hands-on of biopharmaceutical biomanufacturing (and relate to crossover industries).
    • Educate teachers re: local opportunities for STEM biomanufacturing education and training and careers (biomanufacturing career paths).
  • 7. Northeast Biomanufacturing Center Web Site
    • Information available to all
    • Access to global biomanufacturing curricula
    • http://www.biomanufacturing.org
  • 8. RESEARCH & DEVELOPMENT(pre-clinical): Discovery Research OPERATIONS: Process development, Manufacturing & Production QUALITY: Quality Control & Assurance CLINICAL RESEARCH: Clinical Research: Regulatory Affairs Discovery Research Senior Scientist Scientist III,II,I Research Associate Process Development Director Supervisor & Process Development Technician Quality Control (QC) Clinical Research Clinical Research Manager Bioinformatics Scientist Engineer Analyst Programmer Manufacturing & Production Supervisor Associate Technician (Operator) Instrumentation Tech Calibration Technician Facilities Management Manager Facilities Technician Shipper/receiver Chemistry Chemistry QC Analyst QC technician Microbiology Microbiology QC analyst QC Technician Quality Assurance (QA) Documentation Specialist QA Documentation Coordinator Clinical Research Clinical Research Manager Regulatory Affairs Manager Associate Data Manager Business Development Director of Business Development Administration Human resources Safety Manager
    • Careers in red indicate
    • entry level positions
    Entry level positions require an Assoc deg or certificate Higher entry levels require a BS, MS, PhD or Engineering degrees
  • 9. Biomanufacturing Curriculum: National Standard Concepts Covered
    • Math, Biology, Chemistry in Curriculum
      • Measurement
      • Solutions
      • Enzyme reactions
      • Transformation of cells with DNA
      • Forces used in centrifugation, electrophoresis etc.
    • National Academy of Sciences Standards
      • Unit of Math & Science
      • Structure/properties of matter
      • Chemical reactions/conservation of matter
      • Cell structure and functions, heredity
      • Motions and forces
  • 10. Career Tracks: Protein is Cash Introduces
    • Upstream Processing
      • Production of pGLO into protein by transformed cell
    • Downstream Processing
      • Separation of cellular debris and cell supernatant
      • Purification of pGLO protein by Chromatography
    • Quality Control
      • Identification of protein product by electrophoresis
    • Discovery Research
      • How new drugs are discovered
    • Biofuels
  • 11. Biomanufacturing
    • Offers diverse career pathways
    • Hands-on learning of science
    • Understanding of good laboratory practices
    • Stability in career pathways
    • Assistance in obtaining college degrees through company support
    • Rewarding Career
  • 12.  
  • 13. High School Curriculum A Workshop to Instruct Teachers and Students in Biomanufacturing and the Bioeconomy
  • 14. Overview of Protein is Cash
    • http://www.biomanonline.org
  • 15.
    • Metrology
    • Transformation of Bacteria
    • Upstream Processing
    • Downstream Processing
    • Quantitative Analysis
    • Discovery Research
  • 16. Day 1 : Metrology/Instrumentation
    • Activities
    • Calibration of top balance
    • Verification of
    • pipet performance
    • Pipetman Challenge
    • Outcomes
    • Introduction of Good Manu-facturing Practices
    • SOP and documentation
    • Confidence in measurements by instru-
    • mentation
  • 17. Metrology
  • 18. Day 2 Transformation of Bacteria
    • Activities
    • Addition of pGLO plasmid to bacteria in Ca++ solution
    • Heat /shock the mixture
    • Plate cells onto selective Luria broth agar + ampicillin
    • Incubate overnight at 37oC
    • Note: arabinose acts to turn on the gene which starts production of pGLO protein
    • Outcomes
    • Selection of cells by growing on ampicillin plates
    • Only transformed with cells will survive due to amp-r gene
    • Selected colonies will multiply in Luria broth at 37oC overnight
    • Aseptic technique and proper disposal of bacteria
  • 19. Day 2 E.coli Transformation with pGLO plasmid Ampicillin resistance gene (Amp r ) and target gene on bacterial plasmid Bacterial clones Cell division Transformation mixture is plated on to agar plate containing Ampicillin Only E. coli containing plasmid survive on Ampicillin plates
  • 20. Results of Inserting Foreign DNA into an Organism Cells will multiply and produce desired gene product pGlO gene expression vector: Green Fluorescent Protein
  • 21. Day 2: Upstream Processing: Cell Growth and Lysis
    • Activities
    • Transformed cells grown overnight in selected media are separated from media by centrifugation
    • Media is removed and packed cells are lysed
    • Homogenate is centrifuged
    • Supernatant with pGLO protein is retained for
    • downstream processing
    • Outcomes
    • Multiplication of cells
    • Initial separation of
    • fluorescent protein from
    • cell homogenate
  • 22. Day 3: Downstream Processing: Purification of Green Fluorescent Protein by Chromatography
    • Activities
    • Separation of product by Different types of Chromatography
      • Size exclusion
      • Hydrophobic Interaction
      • Cationic Interaction
      • Anionic Interaction
    • Outcomes
    • Fractions with green fluorescent protein will glow and be selected
    • for analysis
    • Understand concept of chromatography as selective interactions of compounds with matrix
  • 23. Isolation and Purification of Green Fluorescent Protein Transformed cells #1 #2 #3 Fraction number Test tubes Courtesy of Bio-Rad
  • 24. Separation of Proteins by Column Chromatograph
    • Separation by size
      • Size exclusion chromatography
    • Separation by hydrophobic characteristics
      • Water loving vs water hating environments
    • Separation by ionic charge
      • Protein has a positive charge & attaches to negative matrix on column: Cation Exchange
      • Protein has a negative charge & attaches to positive matrix on column: Anion Exchange
  • 25. Size Exclusion Chromatography Red molecule =10^6 daltons - Blue molecule = 600 daltons
  • 26. How Size Exclusion Works
    • Molecular size of molecule will separate two or more molecules
    • Large molecules can not go into a bead of a certain size and flows quickly through a column
    • Small molecules enter into a bead and flows slowing through a column.
    • Size of two different molecules are separated
  • 27. Hydrophobic Interaction Chromatography
    • Low salt
    H+ H+ H+ H+ High salt: 2 M (NH 4 ) 2 SO 4 Wash buffer: 1.3 M (NH 4 ) 2 SO 4 Elution buffer: 10 mM Tris Hi Salt
  • 28. Ion Exchange Chromatography
    • Proteins bind to opposite charges on the matrix
    • An example of ion Exchange
    • Addition of increasing
    • Salt/pH should release proteins
  • 29. Cation Exchange vs Anion exchange
    • How proteins are attracted to ion exchangers
  • 30. Day 4: Quality Control
    • Activities
    • Chromatography fractions prepared for electrophoresis
    • Electrophoresis Box is assembled with PAGE gel
    • Samples of chromatography fractions are added to PAGE gel and ran for 30 minutes
    • Gels stained and viewed
    • Outcomes
    • Analysis of protein samples by observation on light box determines protein purity
    • Standard molecular weight markers indicate size of protein
    • Verification of mol wt by comparison with standard proteins and number of proteins in a single sample
  • 31. Quality Control Analysis of Column Fractions
    • Isolated fractions using Ion Exchange Chromatography are then analy
    • Electrophoresis by SDS PAGE of fractions collected
  • 32.  
  • 33. PAGE of pGLO samples after IEX chromatography A B C D E F G H I J A = molecular ladder B = no sample C = IEX #1 fraction column D = I EX # 2 fraction column E -G = IEX wash fractions H = Supernatant I = molecular ladder J = standard GFP 29,000 mol wt 29,000 mol wt
  • 34. Day 5. Discovery Research and FDA approval process
    • Questions
    • Discovery Science in Drug development
    • Good Manufacturing Practices and the FDA
    • Initial Biomanufacturing
    • Process Development
    • Scale up to full production
    • of biologic /clinical trials
    • Answers
    • Visit local biomanufacturing plants
    • Discuss importance of documentation in the workplace ie. SOP and Batch Records
    • Invite industry representatives to speak to the regulated workplace
  • 35. Bio-Rad Partnership
    • Northeast Biomanufacturing Center and Collaborative (NBC 2 ) is partnered with Bio-Rad:
    • Provides teachers with engaging hands-on
    • biomanufacturing education that is easily
    • accessible
    • Introduction to:
      • metrology,
      • production (upstream and downstream processing)
      • quality control biochemistry and clinical trials.
      • These modules can be brought directly into the classroom
  • 36. $ Protein is Cash $ Local Teachers Workshops Biomanonline.org
  • 37. Protein is Cash Workshop Teaching Biomanufacturing in HS
    • Integrated science education: Science Technology Engineering and Math (STEM)
    • Career pathways towards work/school focused on science with thousands of new jobs predicted in the next few years
    • Laboratory based activities = increased interest
      • More exciting ways of introducing concepts
      • Hands-on learning is more inclusive
      • State-of-the-art laboratories
  • 38. HTTP
    • Virtual Chromatography
    • http://www.Atelearning.com/BioChrom
  • 39. Useful Information for Biomanufacturing
    • http://www.Biomanufacturing.org
    • http://www.Biomanonline.org
    • http://www.Bio-link.org
    • http://www.fda.gov
    • Background Resources:
    • “ Development of Biotechnology Curriculum for the Biomanufacturing Industry”, Robert McKown, and George L. Coffman, May/June 2002, Pharmaceutical Engineering pages 1-6.
    • “ Introduction to Biomanufacturing a Global Biomanufacturing Curriculum”
    • Northeast Biomanufacturing Center & Collaborative (NBC2) 2011,
    • publisher :Lulu.com