Fast-Performance Liquid Chromatography.

clatteringlippsΒιοτεχνολογία

5 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

253 εμφανίσεις

Chapter
9

Bringing a Biotechnology

Product to Market


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

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.

H
ow 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
).


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

Vocabulary

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
10
X 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?