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Chapter 9
Learning Outcomes
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
Recovery
Separate the protein from cell debris.
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 same concentration 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 a membrane Load the initial sample loaded onto a column before it is separated via chromatography
1. 2. 3. 4.
When harvesting broth cultures, how are cells separated from the broth? In a column chromatography, what accomplishes the separation of molecules in a mixture? What are the samples called that are collected from a column? What happens during dialysis? Why is dialysis an important technique in protein purification?
Paper Chromatography
Paper chromatography. Molecules separate as they move up the paper. The distance that the molecules travel depends on their size and solubility in the solvent.
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.
Column Chromatography
Gel-Filtration (Size-Exclusion) Chromatography
Gel Filtration Resin. When starting protein purification, technicians sometimes use a gel-filtration (size-exclusion) column first. They know the molecular weight of their protein, so they can often eliminate several contaminant proteins by a quick run through a sizing column.
Ion-Exchange Chromatography
Ion Exchange Resin. Resins are manufactured with ions attached. The ions present a certain degree of positive or negative charge, depending on the buffer pH.
Affinity Chromatography
Affinity Chromatography. Separating molecules based on shape is often done using antibody resin. Antibodies recognize only certain antigens and will bind those and pull them out of solution (fraction #3).
Vocabulary
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 used in chromatography (ie, paper, resin, etc.) through which the molecules of interest move and separate Gel-filtration chromatography a type of column chromatography that separates proteins based on their size using size-exclusion beads; also called size-exclusion chromatography Ion-exchange chromatography a separation technique that separates molecules based on their overall charge at a given pH Affinity chromatography a type of column chromatography that separates proteins based on their shape or attraction to certain types of chromatography resin Hydrophobic-interaction chromatography column chromatography that separates molecules 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
2. 3.
4.
There are two ways to run a column: 1. 2. Allow gravity to draw samples and buffers through the column resin. Use pumps to push a sample and buffers through a column.
Open Column
Also called gravity-flow chromatography
Vocabulary
Open-column chromatography a form of column chromatography that operates by gravity flow Fast-performance liquid chromatography (FPLC) - a type of column chromatography where pumps push buffer and sample through the resin beads at a high rate; used mainly for isolating proteins (purification)
High-performance liquid chromatography (HPLC) a type of column chromatography that uses metal columns that can withstand high pressures; used mainly 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 a high or low pH
1.
A technician wants to quickly determine if an antibody affinity resin will bind a particular protein for purification. Which type of chromatography should he or she use to test the resin? Which instrument, FPLC or HPLC, is used for large-scale protein separations/purifications? Why are spectrophotometers hooked up to most FPLC or HPLC units? You are to dialyze 10 mL of protein extract in PAGE running buffer into sodium monophosphate buffer before running an FPLC ion-exchange column. Into what volume of sodium monophosphate buffer should you place the dialysis bag?
2. 3. 4.
The QC and Quality Assurance (QA) departments monitor the characteristics and performance of the companys products.
Vocabulary
Quality Assurance a department that deals with quality objectives and how they are met and reported internally and externally Investigational New Drug (IND) an application, filed with the FDA for the purpose 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 proposed pharmaceutical Clinical testing another name for clinical trials Double-blind test a type of experiment, often used in clinical trials, in which both the experimenters and test subjects do not know which treatment the subjects receive Placebo an inactive substance that is often used as a negative control in clinical trials
1. 2. 3.
What type of biotechnology product undergoes clinical testing/clinical trials? How many people (subjects) are usually involved in Phases I, II, and III of a clinical trial? In which phase of a clinical trial, Phase I, II, or III, is product safety tested?
Marketing
Advertise and publicize the product to the appropriate 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
Product Applications
Once a product is being synthesized and has been approved, companies look for other applications.
Vocabulary
Proprietary rights confidential knowledge or technology Patent protection the process of securing a patent or the legal rights to an idea or technology
1. 2. 3.
What are some of the reasons that a product in development may not make it to the marketplace? What is covered in an employees proprietary-rights contract? Why must a company gain patent protection on a product?