Transcript Ch6_Presentationx

Identifying a Potential
Biotechnology Product
Chapter 6
Learning Outcomes
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Give examples of biotechnology products derived from plant and
animal sources and discuss the challenges of extracting compounds
Identify the steps in a Comprehensive Product Development Plan and
use it to determine whether a potential biotechnology product is
worth manufacturing
Discuss the types of assays done as potential products move through
process development and identify the additional assays required for
pharmaceutical development
Describe how an ELISA or a Western blot is conducted and what the
results of each assay can reveal
Explain how scientists test the effectiveness of antibiotics and
antimicrobials and discuss the significance of antibiotic resistance.
Describe the role of CHO cells in protein product development
Describe the typical recombinant DNA protein product pipeline,
additional steps required by the FDA for pharmaceutical proteins,
and possible formulations of the final product
6.1 Sources of Potential Products
For thousands of years, people have used plant and animal
organs for many purposes.
Recently, scientists have learned how to use plant and animal
parts as sources of products:
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Pancreas of livestock is used to make insulin.
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Foxglove plant is used to regulate irregular heartbeat.
Harnessing the Potential of Materials Produced in Nature
Sometimes a product is made in very small quantities in nature so it must be produced
Modeling the Research and Development of a Potential Product
Estimating market size
Identifying product sources
Creating a Comprehensive Product Development Plan (CPDP)
1. Does the product meet a critical need? Who will use the product?
2. Is the market large enough to produce sufficient sales? How many customers are
there?
3. Does preliminary data support that the product will work? Will it do what the company
claims?
4. Can patent protection be secured? Can the company prevent other companies from
producing it?
5. Can the company make a profit on the product? How much will it cost to make it? How
much can it be sold for?
Vocabulary
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Amylase – an enzyme that functions to break down the polysaccharide amylose
(plant starch) to the disaccharide maltose
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Assay – a test
6.1 Review Questions
1.
Why are antibiotics important biotechnology products?
2.
What is the function of the enzyme, amylase?
3.
Why might a company be interested in producing amylase
as a product?
4.
Summarize the criteria that a potential product must meet
in a CPDP review.
6.2 The Use of Assays
If a substance is chosen as a potential product,
researchers must be able to test for its presence, activity,
and concentration. The product must be “assayed.”
Other assays are done for potency, toxicity, and stability.
Assays are performed at every step in the development of
a product.
The 24-well plate reveals samples with different amounts of amylase
activity. Amylase breaks down starch to sugar. In the activity assay, as
amylase breaks down starch, the dark color of a starch/iodine mixture
becomes lighter. The lighter the color, the more active the amylase.
Decreasing amounts of protein are indicated by less blue color in the tube.
The right-hand tube is a negative control that contains no protein. The
tubes to the left have increasing concentrations of protein.
Vocabulary
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Activity assay – an experiment designed to show a molecule is
conducting the reaction that is expected
Concentration assay – a test designed to show the amount of
molecule present in a solution
Investigational New Drug (IND) application – a document to
the FDA to allow testing of a new drug or product on humans.
Pharmacodynamic (PD) assay – an experiment designed to
show how a drug is metabolized (processed) in the body
Potency assay – an experiment designed to show the
biochemical effect of a drug on the body
Toxicology assay – an experiment designed to find what
quantities of a drug are toxic to cells, tissues, and model
organisms
Stability assay – an experiment designed to determine the
conditions that affect the shelf life of a drug
6.2 Review Questions
1.
What kind of assay would use Bradford reagent in the test?
2.
For what purpose would a technician use an ELISA?
3.
What does a stability assay measure?
4.
In a large company, which department would have several
employees developing and conducting assays?
6.3 Enzyme Linked Immunosorbent Assay (ELISA)
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One of the most important and frequently used molecular
assays is the ELISA.
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An ELISA is very specific and will recognize a single type of
protein or other antigenic molecule in a mixture of others.
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ELISA utilizes two important phenomenon:
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1) antibody antigen specificity to recognize only very
specific molecules
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2) enzyme activity on colorimetric reagents for
visualization purposes.
In an ELISA, the goal is to recognize the antigen and measure
its concentration.
Vocabulary
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Direct ELISA – an ELISA where a primary antibody
linked with an enzyme recognizes an antigen and
indicates its presence with a colorimetric reaction
Indirect ELISA – an ELISA where a primary antibody
binds to an antigen and then a secondary antibody
linked with an enzyme recognizes the primary
antibody—the antigen presence and concentration is
indicated by the degree of a colorimetric reaction
6.3 Review Questions
1.
Explain how antibodies and enzymes are used in ELISAs.
2.
How can a technician know that an antigen is present during
an ELISA?
3.
How can a technician know the concentration of an antigen in
an ELISA?
4.
What is the difference between a direct and an indirect ELISA?
6.4 Western Blots
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In a Western blot, samples are run on a PAGE gel and then the
protein bands are transferred to a blotting membrane (PVDF or
nitrocellulose).
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The visualization of a Western blot can confirm the presence of
a particular protein at very low concentrations.
Western Blot Gel/Transfer Membrane Setup Diagram.
During a Western blot, electrical current carries protein bands from
the PAGE gel to the blot transfer membrane.
Vocabulary
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PVDF– polyvinylidene fluoride, a high molecular weight fluorocarbon
with dielectric properties that make it suitable for Western blots because
it is attractive to charged proteins
Nitrocellulose – a modified cellulose molecule used to make paper
membrane for blots of nucleic acids and proteins
Antiseptic – antimicrobial solution, such as alcohol or iodine, that is
used to clean surfaces
6.4 Review Questions
1.
What kind of molecule is being blot during a Western blot?
2.
What causes molecules to move from a gel to a membrane in a
Western blot?
3.
How are blotted molecules visualized during a Western blot?
4.
For what purpose are Western blots used in industry?
6.5 Looking for New Products in Nature
Herbal remedies from the rainforest
Vocabulary
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Herbal remedies – the products developed from
plants that exhibit or are thought to exhibit some
medicinal property
Antimicrobial – a substance that kills or slows the
growth of one or more microorganisms
Antiseptic – an antimicrobial solution, such as alcohol
or iodine, that is used to clean surfaces
6.5 Review Questions
1.
From where do scientists expect that most of the remaining
naturally occurring biotechnology products will come?
2.
How can a technician know if a certain type of bacteria is
sensitive to an antimicrobial substance?
3.
List a few herbal products that claim to have therapeutic value
against depression.
4.
How can molecules be extracted from plant samples for testing
purposes?
6.6 Producing Recombinant DNA (rDNA) Protein Products
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rDNA was first produced in the mid 1970s.
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Recombinant refers to the process of
combining DNA from two sources.
Vocabulary
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Transfection – the genetic engineering, or transformation of mammalian cell lines
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Formulation – the form of a product, as in tablet, powder, injectable liquid, etc.
6.6 Review Questions
1.
What are CHO cells and what are they used for?
2.
How long does it take to develop, test, and market a typical
rDNA protein product?
3.
What does GMO stand for and what does it cover?
4.
Biotechnology products must be formulated before they can be
marketed. Name two formulations for a pharmaceutical product
other than tablet form.
Questions and Comments?