Chapter 9

1. Bringing a Biotechnology Product to Market Chapter 9

2. 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

3. 9.1 Harvesting a Protein Product The method of harvesting a protein from cloned cells depends on whether that protein is found within the cell or outside the cell. Recovery Separate the protein from cell debris. How a Column Works

4. 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

5. 9.1 Review Questions • 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?

6. 9.2 Using Chromatography to Study and Separate Molecules 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.

7. 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. 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.

9. 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.

10. 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).

11. 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 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 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

12. 9.2 Review Questions • What is the solid phase for each of the following types of chromatography? • paper chromatography • thin-layer chromatography • gel-filtration chromatography • ion-exchange chromatography • affinity chromatography • If a molecule is the smallest in a mixture, will it be the first or last molecule to come off a size-exclusion column? • Diethylaminoethyl (DEAE) sepharose is a type of ion-exchange resin. At a pH of 7.5, it has a positive charge. What would be expected if a sample containing one positively charged protein and one negatively charged protein were put on a DEAE column? Where should the proteins end up? • What is the value of a fraction collector?

13. 9.3 Column Chromatography: An Expanded Discussion • There are two ways to run a column: • Allow gravity to draw samples and buffers through the column resin. • Use pumps to push a sample and buffers through a column.

14. Open Column Also called gravity-flow chromatography Fast-Performance Liquid Chromatography (FPLC) Fast-Performance Liquid Chromatography. Pumps push the buffer or sample through tubing, into and through the column. As fractions come off the column, they are run through a spectrophotometer that determines the protein concentration of the sample.

15. 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 positive or negative charges at a given pH.

16. Buffers Used in Column Chromatography Dialysis Buffer Exchange. Typically, dialysis is conducted using 10X the volume of the buffer outside the bag as that inside the bag. Also, the buffer is changed after several hours. This ensures the complete exchange of buffers. Sometimes the volume of the sample increases substantially from the influx of buffer. If this happens, the sample can be concentrated using concentrators or centrifuge filters.

17. 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. 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

19. 9.3 Review Questions • 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?

20. 9.4 Product Quality Control The QC and Quality Assurance (QA) departments monitor the characteristics and performance of the company’s products.

21. 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

22. 9.4 Review Questions • 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?

23. 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 company may hold proprietary rights.

24. 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 product’s target population • Timing for FDA approval of competitive products • Rate of market penetration for competitive products

25. Proprietary/Patent Rights, and Community and Government Regulations Intellectual theft Strong patent protection Product Applications Once a product is being synthesized and has been approved, companies look for other applications.

26. 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. 9.5 Review Questions • What are some of the reasons that a product in development may not make it to the marketplace? • What is covered in an “employee’s proprietary-rights contract”? • Why must a company gain patent protection on a product?

28. Questions and Comments?