Biotech 201:
The Technology That Drives
Biotech
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Objectives
At the end of this webinar you will be able to:
•
define recombinant DNA
•
explain the steps used to make recombinant DNA
•
understand the uses of recombinant DNA
•
understand how recombinant DNA is used to produce recombinant proteins
•
understand the uses of recombinant proteins
•
explain why one cell is chosen over another e.g. bacterial cell vs. mammalian cell
•
choose the best DNA transfer method, based on your cell and plasmid, of
•
write out the key steps involved in a biomanufacturing campaign
•
understand the uses of cell banks, how they are created, and how they are used
•
explain the basics of how the scale up process works
•
understand the challenges of product purification
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Cells Manufacture
energy
information
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Restriction Enzymes
Each restriction enzyme recognizes a specific sequence
in the DNA and then cuts within the site:
GGTTG
AATTCGGCTTACCCC
CCAACTTAA
GCCGAATGGGG
EcoRI
SmaI
GGGAGCGCTGC
GGCCGCCCTT
CCCTCGCGACGCCGG
CGGGAA
NotI
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Recombinant DNA
Bacterial DNA
Human DNA
DNA Ligase
Restriction
Enzyme
Recombinant DNA
Ligation: two different DNA pieces can be recombined or “ligated” together
Vector: vehicle to carry genetic material to new cell
Plasmid: common type of vector, circular piece of DNA
Each vector is chosen for specific characteristics:
• size of DNA fragment
• Linked selection marker (antibiotic resistance or color)
Application: Transfer a gene from one organism to another, often for the purpose of
protein production
.
bacterium
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Making A Plasmid
Plasmid
Antibiotic
resistance gene
Gene of
Interest
Restriction
enzymes
DNA ligase
Recombinant
Plasmid
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Making Recombinant Proteins In Bacteria
Recombinant
Plasmid
Add Antibiotic:
Bacterium
= Antibiotic Resistance
Gene
Recombinant Protein
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Making Recombinant Proteins In
Mammalian Cells
Recombinant
DNA
CHO
Epogen
Plasmid
Human
Erythropoietin
Gene
CHO Contains
Human
Erythropoietin
Gene
CHO Cells Grow
Human
Erythropoietin
Protein
Product
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Transfer Of DNA
Plasmids carrying the new gene can be put into:
or
Bacterial Cells
Eukaryotic Cells
Methods:
Chemical
Electroporation
Liposomes
Microinjection
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Production Options:
Mammalian vs. Bacterial Cells
Bacterial
Mammalian
• Grow quickly – divide every 20-30 minutes
• Production campaign can be completed in days
• Growth media is relatively cheap
• Less sensitive to slight changes in environment
• Can only produce simple proteins
• Divide on average once a day
• Production campaign typically takes weeks
• Expensive, specialized growth media
• Sensitive to slight environmental changes
• Capable of producing highly complex proteins
• Proper folding
• Post-translational modifications
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Recombinant Proteins In Healthcare
Antibodies Against
Autoimmune Disease
Or Disease Proteins
Replacement or
Supplement
Regulatory Proteins
Erythropoietin (Epogen) Amgen
Non-Hodgkins Lymphoma
(Rituxan) Biogen Idec
Vaccines
Recombinant HPV protein
(Gardasil) Merck
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Engineered Mammalian Cell Uses
Freeze
Protein Expression
• Soluble
• Proper Folding
• “More Human”
Research Model
• Gene Expression
• Cell Function
• Disease Model
Healthcare
• Drug Discovery
• Efficacy Testing
• Safety Testing
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Biologics Production Overview
3. Chromosome
4. Gene of Interest
9. Insertion
1. Biochemicals
2. Tissue
7. Cut Plasmid
8. Recombinant
Plasmid
6. Plasmid
10. Replication
5. Microorganism
13. Pilot-Scale
Bioreactor
14. Industrial Scale Operation
Source: Bioprocess Engineering Principles
12. Bench Top
Bioreactor
15. Product Recovery
11. Small Scale
Culture
16. Packaging & Marketing
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Cell Banking
Why make a cell bank?
Assures identical product by using identical starting material
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Cell Bank Production
Culture of newly
constructed
production cell line
Establish master
cell bank (MCB)
1 2 3
4 5
1
1 2
3 4 5
Establish working cell bank (WCB)
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Scale-Up And Manufacturing Process
Cell Culture Seeding
Culture Scale-Up
Stock Culture
Shake Flask
Quality control monitoring:
• cell viability and concentration
• product concentration and activity
• optimal growth conditions
Bench Top Bioreactor
Production Bioreactor
Culture Fluid
In accordance with cGMP
Source: Principles of Fermentation Technology
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Harvesting & Purification Process
Cell Culture Fluid
Protein
mixture
Cell Separation
Chromatography used to
separate proteins based on:
• charge
• structure
• size
Column
Elution
profile
Cell-Free Supernatant
Matrix
Product Extraction
UV Detector
Product Purification
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Summary
The Technology That Drives Biotech
Recombinant DNA technology is used to transfer a gene from one organism
to another
A bacterial or mammalian cell can be used to produce large quantities of a
human protein
The process of using living cells to produce a therapeutic protein is called
biomanufacturing
Genetically engineered cell lines are frozen and stored for later use, and are
critically important to a company’s manufacturing production
“Scale-up” refers to the series of steps that must be taken in order to grow a
large volume of cells for biomanufacturing
Product purification takes advantage of chemical and physical differences in
proteins to purify the desired product
Copyright © 2012 Biotech Primer Inc. All rights reserved.
Thank You For Participating
Want to learn more?
Go to www.biotechprimerinc.com to see a listing of
upcoming classes or contact Stacey Franklin at
443.798.2385 [email protected] to discuss
customized in-house classes.
Copyright © 2012 Biotech Primer Inc. All rights reserved.