Biotech_chapter_9.ppt - PowerPoint Presentation

							Bringing a Biotechnology
Product to Market

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
     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
            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
     9.1 Review Questions


1.   When harvesting broth cultures, how are cells separated from the
     broth?
2.   In a column chromatography, what accomplishes the separation of
     molecules 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?
    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.
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
         9.2 Review Questions


1.   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
2.   If a molecule is the smallest in a mixture, will it be the first or last molecule
     to come off a size-exclusion column?
3.   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?
4.   What is the value of a fraction collector?
9.3 Column Chromatography: An Expanded Discussion



 There are two ways to run a column:
 1.   Allow gravity to draw samples and buffers through
      the column resin.
 2.   Use pumps to push a sample and buffers through a
      column.
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.
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.
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.
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
    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
     9.3 Review Questions


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?
2.   Which instrument, FPLC or HPLC, is used for large-scale protein
     separations/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 into
     sodium monophosphate buffer before running an FPLC ion-exchange
     column. Into what volume of sodium monophosphate buffer should you
     place the dialysis bag?
9.4 Product Quality Control




The QC and Quality Assurance (QA) departments monitor the
characteristics and performance of the company’s 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
     9.4 Review Questions



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 safety
     tested?
      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.
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
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.
Vocabulary


•   Proprietary rights – confidential knowledge or technology
•   Patent protection – the process of securing a patent or the legal rights to an
    idea or technology
     9.5 Review Questions



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 “employee’s proprietary-rights contract”?
3.    Why must a company gain patent protection on a product?
Questions and Comments?