Biotech Chapter 9

7/28/2019 Biotech Chapter 9

1/28

Chapter 9

Bringing a BiotechnologyProduct to Market


2/28

Compare and contrast the methods of harvesting intracellular and

extracellular proteins Define chromatography and distinguish between paper, thin-layer, and

column chromatography, giving examples of each procedure Discuss the variables used to optimize column chromatography Explain how product quality is maintained for key types of

biotechnology and pharmaceutical products

Describe the clinical testing process for pharmaceuticals Discuss the final marketing and sales considerations in bringing a

product to market

Learning Outcomes


3/28

9.1 Harvesting a Protein Product

The method of harvesting a protein from cloned cells depends onwhether that protein is found within the cell or outside the cell.

Recovery

Separate the protein fromcell debris.

How a Column Works


4/28

Vocabulary Quality Control (QC) a department in a company that monitors the quality of a product and all the

instruments and reagents associated with it Harvesting extracting protein from a cell culture Intracellular within the cell Extracellular outside the cell Sonication the use of high frequency sound waves to break open cells

Recovery the retrieval of a protein from broth, cells, or cell fragments Purification the process of eliminating impurities from a sample; in protein purification, it is the separation

of other proteins from the desired protein Column chromatography a separation technique in which a sample is passed through a column packed

with resin (beads); the resin beads are selected based on their ability to separate molecules based on size,shape, charge, or chemical nature

Gravity-flow columns column chromatography that uses gravity to force a sample through resin beads Pressure-pumped columns a column chromatography apparatus that uses pressure to force a sample

through the resin beads Frit the membrane at the base of a chromatographic column that holds the resin in place Fraction a sample collected as buffer flows over the resin beads of a column Dialysis process in which a sample is placed in a membrane with pores of a specified diameter, and

molecules, smaller in size that the pore size, move into and out of the membrane until they are at the sameconcentration on each side of the membrane; used for buffer exchange and as a purification technique

Diafiltration a filtering process by which some molecules in a sample move out of a solution as it passes amembrane

Load the initial sample loaded onto a column before it is separated via chromatography


5/28

9.1 Review Questions

1. When harvesting broth cultures, how are cells separated from thebroth?

2. In a column chromatography, what accomplishes the separation ofmolecules in a mixture?

3. What are the samples called that are collected from a column?

4. What happens during dialysis? Why is dialysis an important technique in

protein purification?


6/28

9.2 Using Chromatography to Study and Separate Molecules

Paper Chromatography

Paper chromatography. Molecules separate as they move up the paper. Thedistance that the molecules travel depends on their size and solubility in the solvent.


7/28

Thin-Layer Chromatography

Thin-layer chromatography. Molecules separate as they move through the silica gel.Thin-layer chromatography is used to separate small molecules, such as amino acids.


8/28

Column Chromatography

Gel-Filtration (Size-Exclusion) Chromatography

Gel Filtration Resin. Whenstarting protein purification,technicians sometimes use agel-filtration (size-exclusion)

column first. They know themolecular weight of theirprotein, so they can ofteneliminate several contaminantproteins by a quick runthrough a sizing column.


9/28

Ion-Exchange Chromatography

Ion Exchange Resin.Resins are manufacturedwith ions attached. The ionspresent a certain degree ofpositive or negative charge,depending on the buffer pH.


10/28

Affinity Chromatography

AffinityChromatography.Separating molecules basedon shape is often doneusing antibody resin.

Antibodies recognize only

certain antigens and willbind those and pull themout of solution (fraction #3).


11/28

Paper chromatography a form of chromatography that uses filter paper as the solid phase,and allows molecules to separate based on size or solubility in a solvent

Thin-layer chromatography a separation technique that involves the separation of small

molecules as they move through a silica gel Chromatograph the medium usedin chromatography (ie, paper, resin, etc.) through which

the molecules of interest move and separate

Gel-filtration chromatography a type of column chromatography that separates proteinsbased on their size using size-exclusion beads; also called size-exclusion chromatography

Ion-exchange chromatography a separation technique that separates molecules based ontheir overall charge at a given pH

Affinity chromatography a type of column chromatography that separates proteins based ontheir shape or attraction to certain types of chromatography resin

Hydrophobic-interaction chromatography column chromatography that separatesmolecules based on their hydrophobicity (aversion to water)

Elution when a protein or nucleic acid is released from column chromatography resin

Cation exchange a form of ion-exchange chromatography in which positively charged ions(anions) are removed by a positively charged resin

Vocabulary


12/28


1. What is the solid phase for each of the following types of chromatography?paper chromatography

thin-layer chromatographygel-filtration chromatographyion-exchange chromatographyaffinity chromatography

2. If a molecule is the smallest in a mixture, will it be the first or last moleculeto come off a size-exclusion column?

3. Diethylaminoethyl (DEAE) sepharose is a type of ion-exchange resin. At a pHof 7.5, it has a positive charge. What would be expected if a samplecontaining one positively charged protein and one negatively charged proteinwere put on a DEAE column? Where should the proteins end up?

4. What is the value of a fraction collector?


13/28

9.3 Column Chromatography: An Expanded Discussion

There are two ways to run a column:

1. Allow gravity to draw samples and buffers throughthe column resin.

2. Use pumps to push a sample and buffers through acolumn.


14/28

Open Column

Also called gravity-flow

chromatography

Fast-Performance LiquidChromatography (FPLC)

Fast-Performance Liquid Chromatography.Pumps push the buffer or sample throughtubing, into and through the column. Asfractions come off the column, they are runthrough a spectrophotometer that determinesthe protein concentration of the sample.


15/28

High-Performance Liquid Chromatography (HPLC)

Greatly improved ability to separate, purify, identify, and

qualify samples.

Resins Used in Column Chromatography

There are several types of resins available.

For ion-exchange chromatography, resins have either positiveor negative charges at a given pH.


16/28

Buffers Used in Column Chromatography

Dialysis Buffer Exchange. Typically,dialysis is conducted using 10X thevolume of the buffer outside the bag asthat inside the bag. Also, the buffer ischanged after several hours. This ensuresthe complete exchange of buffers.

Sometimes the volume of the sampleincreases substantially from the influx ofbuffer. If this happens, the sample can beconcentrated using concentrators orcentrifuge filters.


17/28

Resin Bed Versus Sample Concentration

The amount of resin must be sufficient to interact with the sample

Best conditions are discovered through trial and error


18/28

Vocabulary

Open-column chromatography a form of column chromatography that operatesby gravity flow

Fast-performance liquid chromatography (FPLC) - a type of column

chromatography where pumps push buffer and sample through the resin beads at ahigh rate; used mainly for isolating proteins (purification)

High-performance liquid chromatography (HPLC) a type of columnchromatography that uses metal columns that can withstand high pressures; usedmainly for identification or quantification of a molecule

Equilibration buffer a buffer used in column chromatography to set the charges

on the beads or to wash the column Elution buffer the buffer used to detach a protein or nucleic acid from

chromatography resin; generally contains either a high salt concentration or has ahigh or low pH


19/28


1. A technician wants to quickly determine if an antibody affinity resin willbind a particular protein for purification. Which type of chromatography

should he or she use to test the resin?

2. Which instrument, FPLC or HPLC, is used for large-scale proteinseparations/purifications?

3. Why are spectrophotometers hooked up to most FPLC or HPLC units?

4. You are to dialyze 10 mL of protein extract in PAGE running buffer intosodium monophosphate buffer before running an FPLC ion-exchangecolumn. Into what volume of sodium monophosphate buffer should youplace the dialysis bag?


20/28

9.4 Product Quality Control

The QC and Quality Assurance (QA) departments monitor thecharacteristics and performance of the companys products.


21/28

Vocabulary

Quality Assurance a department that deals with quality objectives and howthey are met and reported internally and externally

Investigational New Drug (IND) an application, filed with the FDA for thepurpose of testing and marketing a product, that describes the structure,specific function, manufacturing process, purification process, preclinical(animal) testing, formulation, and specific application of a proposedpharmaceutical

Clinical testing another name for clinical trials

Double-blind test a type of experiment, often used in clinical trials, in whichboth the experimenters and test subjects do not know which treatment thesubjects receive

Placebo an inactive substance that is often used as a negative control inclinical trials


22/28


1. What type of biotechnology product undergoes clinical

testing/clinical trials?2. How many people (subjects) are usually involved in Phases I, II,

and III of a clinical trial?

3. In which phase of a clinical trial, Phase I, II, or III, is product safetytested?


23/28

9.5 Marketing and Sales

Bringing a Product to Market

Some factors that may impede a product reaching the marketplace:

A product may be found to be ineffective during preclinical or clinical trials.

During testing, a product may be shown to have harmful side effects.

Production may turn out to be uneconomical.

A product may fail to receive necessary regulatory approvals, such as from

the FDA.

Competing products may already control a large portion of the market.

Patent protection for the product may be unobtainable, or another companymay hold proprietary rights.


24/28

Marketing

Advertise and publicize the product to theappropriate audience

Product Sales

Can be affected by:

Effectiveness of the marketing team

Pricing decisions made by the company Degree of patent protection afforded the product

Use of alternative therapies or products for the products target population

Timing for FDA approval of competitive products

Rate of market penetration for competitive products


25/28

Proprietary/Patent Rights, and Communityand Government Regulations

Intellectual theft

Strong patent protection

Product Applications

Once a product is being synthesized and has beenapproved, companies look for other applications.


26/28

Vocabulary

Proprietary rights confidential knowledge or technology

Patent protection the process of securing a patent or the legal rights to an

idea or technology


27/28


1. What are some of the reasons that a product in development may

not make it to the marketplace?2. What is covered in an employees proprietary-rights contract?

3. Why must a company gain patent protection on a product?


28/28

Questions and Comments?

Biotech Chapter 9

Documents

Transcript of Biotech Chapter 9