Bringing a Biotechnology
Product to Market
Chapter 9
Learning Outcomes
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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
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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
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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
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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?
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
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Open-column chromatography – a form of column chromatography that operates
by gravity flow
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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)
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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
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Equilibration buffer – a buffer used in column chromatography to set the charges
on the beads or to wash the column
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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
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Quality Assurance – a department that deals with quality objectives and how
they are met and reported internally and externally
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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
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Clinical testing – another name for clinical trials
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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
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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:
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A product may be found to be ineffective during preclinical or clinical trials.
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During testing, a product may be shown to have harmful side effects.
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Production may turn out to be uneconomical.
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A product may fail to receive necessary regulatory approvals, such as from
the FDA.
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Competing products may already control a large portion of the market.
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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
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Proprietary rights – confidential knowledge or technology
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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?