Identifying a Identifying a Potential Potential

Biotechnology Biotechnology ProductProductChapter 6Chapter 6

ObjectivesObjectives

Describe the CPDP process.Describe the CPDP process. List and describe the different assays List and describe the different assays

that can be performed on proteins.that can be performed on proteins. Describe the processes that can used Describe the processes that can used

to isolate molecules of interest from to isolate molecules of interest from plant cells.plant cells.

Describe how plant proteins of Describe how plant proteins of interest can be mass produced by interest can be mass produced by cells.cells.

Is it worth pursing a Is it worth pursing a product or not?product or not?

Biotechnology products can come Biotechnology products can come from a variety of sources.from a variety of sources.

Decision needs to be made as to Decision needs to be made as to whether it is worth pursuing the whether it is worth pursuing the development and sale of a product.development and sale of a product.

Many companies use a CPDP Many companies use a CPDP strategy.strategy. Comprehensive Product Development Comprehensive Product Development

PlanPlan

Is it worth pursing a Is it worth pursing a product or not?product or not?

CPDPCPDP1.1. Does the product meet a critical need? Who will use Does the product meet a critical need? Who will use

the product?the product?2.2. Is the market large enough to produce sufficient Is the market large enough to produce sufficient

sales? How many customers are there?sales? How many customers are there?3.3. Does the preliminary data support that the product Does the preliminary data support that the product

will work? Will it do what the company claims?will work? Will it do what the company claims?4.4. Can patent protection be secured? Can the Can patent protection be secured? Can the

company prevent other companies from producing company prevent other companies from producing it?it?

5.5. Can the company make a profit on the product? Can the company make a profit on the product? How much will it cost to make? How much can it be How much will it cost to make? How much can it be sold for?sold for?

I.e. common cold vaccineI.e. common cold vaccine AmylaseAmylase

Verifying what you have.Verifying what you have.

A majority of the biotechnology A majority of the biotechnology products produced are proteins.products produced are proteins.

Must be able to determine:Must be able to determine: Is product present?Is product present? What is the overall activity of the product?What is the overall activity of the product? Concentration of product.Concentration of product.

Difficulty in working with proteins is Difficulty in working with proteins is that they are colorless and nonvisible that they are colorless and nonvisible to the naked eye.to the naked eye.

AssaysAssays(Tests)(Tests)

1. Activity assays1. Activity assays 2. Concentration assays2. Concentration assays

Bradford protein reagent or Bradford protein reagent or bicinchoninic acid (BCA)bicinchoninic acid (BCA)

Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA)(ELISA)

Verifying what you have.Verifying what you have.

Generally assays, Generally assays, meaning test, are meaning test, are done when done when working with working with proteins.proteins.

Activity assayActivity assay Indicate presenceIndicate presence Indicate whether Indicate whether

protein is working protein is working or notor not

Lighter color…..more active amylase

Verifying what you have.Verifying what you have. Concentration assayConcentration assay

Amount/unit volume of a Amount/unit volume of a protein protein

By eye or spectrophotometerBy eye or spectrophotometer Bradford protein reagent or Bradford protein reagent or

bicinchoninic acid (BCA)bicinchoninic acid (BCA) Nonspecific in its actionNonspecific in its action

ELISAELISA Enzyme-linked immunosorbent Enzyme-linked immunosorbent

assayassay Very specificVery specific Unitize antigen-antibody specifityUnitize antigen-antibody specifity

An enzyme attached to an antibody An enzyme attached to an antibody binds to a specific protein (antigen)binds to a specific protein (antigen)

Causes a color change that can be Causes a color change that can be meas. When specific substrate is meas. When specific substrate is addedadded

Decreasing amount of protein…..indicated by less blue color in tube

Negative

0.01, 0.1, 1mg/mL

The darker the color…..higher conc. Of protein

Verifying what you have.Verifying what you have. Dealing with pharmaceuticalsDealing with pharmaceuticals

The stringency of verifying what you have, that it The stringency of verifying what you have, that it is safe, and that it works are much higher.is safe, and that it works are much higher.

In addition to activity and conc. In addition to activity and conc. PK assayPK assay

Pharmacokinetic (amount of activity of protein in humans Pharmacokinetic (amount of activity of protein in humans and other test organisms)and other test organisms)

PD assayPD assay Pharmacodynamic (length of activity)Pharmacodynamic (length of activity)

Additional assays developed and carried out to Additional assays developed and carried out to show potency, toxicology, and stability of drug.show potency, toxicology, and stability of drug.

Potency: how dosage of a drug affects its activity and how Potency: how dosage of a drug affects its activity and how long stays in bodylong stays in body

Toxicology: what quantities of drug toxic to cells, tissues, Toxicology: what quantities of drug toxic to cells, tissues, and model organismand model organism

Stability: shelf life, and storage conditionsStability: shelf life, and storage conditions Assay Services and Quality Control Department.Assay Services and Quality Control Department.

Focus on Plant Proteins as Focus on Plant Proteins as Possible ProductsPossible Products

Extracting Protein Molecules from CellsExtracting Protein Molecules from Cells Most molecules of interest are found within Most molecules of interest are found within

the cell.the cell. Problems encountered when attempting to Problems encountered when attempting to

isolate RNA, DNA, or proteins from plant isolate RNA, DNA, or proteins from plant cells.cells.

Plant material is dense and can be quiet woody.Plant material is dense and can be quiet woody. Cells that make up plants have cell walls that are Cells that make up plants have cell walls that are

very thick.very thick. Plant material broken into smaller Plant material broken into smaller

components to increase surface area for components to increase surface area for solvents to work on.solvents to work on.

Focus on Plant Proteins as Focus on Plant Proteins as Possible ProductsPossible Products

Removing or Weakening Cell Removing or Weakening Cell WallsWalls

Enzymatic removal of cell wallEnzymatic removal of cell wall Major components of cell wall are Major components of cell wall are

cellulose and pectin.cellulose and pectin. Cellulase and pectinase enzymes Cellulase and pectinase enzymes

degrade cellulose and pectin.degrade cellulose and pectin. Once cell wall removed protoplast Once cell wall removed protoplast

forms.forms. Plant cell with only a plasma (cell) Plant cell with only a plasma (cell)

membrane.membrane. Homogenization of plant cellsHomogenization of plant cells

Crushing action that results in release Crushing action that results in release of intracellular contents.of intracellular contents.

I.e. TissuemizerI.e. Tissuemizer Works like blenderWorks like blender Grinds tissues without causing foamGrinds tissues without causing foam

Focus on Plant Proteins as Focus on Plant Proteins as Possible ProductsPossible Products

Removing or Weakening Cell WallsRemoving or Weakening Cell Walls Freeze factureFreeze facture

Cells flash frozen with liquid nitrogen or dry iceCells flash frozen with liquid nitrogen or dry ice Crushed using a mortar and pestle Crushed using a mortar and pestle

Results in maceration of cellsResults in maceration of cells

Isolating ProteinsIsolating Proteins Cell contents placed in a neutral solution and Cell contents placed in a neutral solution and

proteins extracted.proteins extracted. i.e. potassium phosphate bufferi.e. potassium phosphate buffer

Other extraction methods that can be used Other extraction methods that can be used are acetone or ammonium sulfate.are acetone or ammonium sulfate.

Once protein(s) isolated assays are generally Once protein(s) isolated assays are generally run.run.

Focus on Plant Proteins as Focus on Plant Proteins as Possible ProductsPossible Products

Once a particular protein has been Once a particular protein has been identified a possible way to make more identified a possible way to make more of it is to produce rDNA.of it is to produce rDNA.

Cells can be transformed with the rDNA Cells can be transformed with the rDNA and begin the process of making the and begin the process of making the protein.protein.

Homework Homework

Review questions page 168Review questions page 168 Questions 1, 2, 3, and 4Questions 1, 2, 3, and 4

Think like biotech page 180Think like biotech page 180 Questions 3, 5, 6, 7, and 9Questions 3, 5, 6, 7, and 9

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