Kurtz biomanufacturing

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

$Protein is Cash$ Local Teachers WorkshopsBiomanonline.org

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

HTTP

Virtual Chromatographyhttp://www.Atelearning.com/BioChrom

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