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Protein is Cash:State of the Art Curriculum to Support the
Bioeconomy
Mary Jane Kurtz, Ph.D NBC2 [email protected]
Basis of the Bioeconomy
Central Dogma: DNA RNA Protein
Discovery Research (DNA Centric) Process Development and Biomanufacturing
(Protein Centric)
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
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
NBC2 Products
The NBC2 has worked for six years with both industry and educators to create biomanufacturing curriculum materials for multiple audiences, utilizing the latest in pedagogical technology.
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).
Northeast Biomanufacturing Center Web Site
Information available to all Access to global biomanufacturing curricula
http://www.biomanufacturing.org
RESEARCH &DEVELOPMENT(pre-clinical):Discovery Research
OPERATIONS:
Process development, Manufacturing& Production
QUALITY:
Quality Control & Assurance
CLINICAL RESEARCH:Clinical Research:RegulatoryAffairs
Discovery Research
Senior ScientistScientist III,II,IResearch Associate
ProcessDevelopmentDirector Supervisor &Process Development Technician
Quality Control (QC)
Clinical Research
Clinical Research Manager
BioinformaticsScientistEngineerAnalystProgrammer
Manufacturing& ProductionSupervisorAssociateTechnician (Operator)Instrumentation TechCalibration TechnicianFacilities ManagementManagerFacilities TechnicianShipper/receiver
ChemistryChemistry QCAnalystQC technicianMicrobiologyMicrobiologyQC analystQC TechnicianQuality Assurance (QA)DocumentationSpecialistQA Documentation Coordinator
Clinical ResearchClinical Research ManagerRegulatory AffairsManagerAssociateData ManagerBusiness DevelopmentDirector of Business DevelopmentAdministrationHuman resourcesSafety 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
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
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
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
High School Curriculum
A Workshop to Instruct Teachers and Students in
Biomanufacturing and the Bioeconomy
Overview of Protein is Cash
http://www.biomanonline.org
1. Metrology2. Transformation of Bacteria3. Upstream Processing4. Downstream Processing5. Quantitative Analysis6. Discovery Research
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
Metrology
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
Day 2 E.coli Transformation with pGLO plasmid
Ampicillin resistance gene (Ampr) and target gene on bacterial plasmid
Bacterial clones
Cell division
Transformation mixture is platedon to agar plate containing Ampicillin
Only E. coli containing plasmidsurvive on Ampicillin plates
Results of Inserting Foreign DNA into an Organism
Cells will multiply and produce desired gene product
pGlO gene expression vector: Green Fluorescent Protein
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
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
Isolation and Purification of Green Fluorescent Protein
Transformed cells
#1 #2 #3Fraction number
Test tubes
Courtesy of Bio-Rad
Separation of Proteinsby 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
Size Exclusion Chromatography
Red molecule =10^6 daltons - Blue molecule = 600 daltons
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
Hydrophobic Interaction Chromatography
Low salt
H+H+ H+
H+
High salt:
2 M (NH4)2SO4
Wash buffer:
1.3 M
(NH4)2SO4
Elution buffer:10 mM Tris
Hi Salt
Ion Exchange Chromatography
• Proteins bind to opposite charges on the matrix
• An example of ion Exchange
• Addition of increasing
Salt/pH should release proteins
Cation Exchange vs Anion exchange
How proteins are attracted to ion exchangers
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
Quality ControlAnalysis of Column Fractions
Isolated fractions using Ion Exchange Chromatography are then analy
Electrophoresis by SDS PAGE of fractions collected
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 columnD = I EX # 2 fraction columnE -G = IEX wash fractions
H = Supernatant
I = molecular ladder
J = standard GFP
29,000 mol wt
29,000 mol wt
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
Bio-Rad Partnership
Northeast Biomanufacturing Center and Collaborative (NBC2) 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
Protein is Cash WorkshopTeaching 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
Useful Information for Biomanufacturing
http://www.Biomanufacturing.org http://www.Biomanonline.org http://www.Bio-link.org http://www.fda.govBackground 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
