Chapter 9 Flashcards
what is the function of ion-exchange chromatography?
separates molecules based on their overall charge at a given pH
How does ion-exchange chromatography work?
Resin beads are given a certain charge through a certain pH, when the sample is running through the chromatography, the ones with the opposite charge will attach to the beads and the other charge will wash away. The ones that stay attached get eluted off by the elution buffer which contains a high salt concentration, or it has a high or low pH, then it is collected.
All fractions are visualized through a PAGE
What is used so that technicians do not have to stand for hours changing collection tubes every
few minutes
fraction collectors
Why would a protein’s charge and behavior change in an ion exchange chromatography?
bc of the pH of the solution in the column
cation exchange
the molecule of interest is a positively charged protein,
then negatively charged resin is used.
anion exchange
protein of interest is negatively charged, then positively charged resin is used.
What is affinity-column chromatography based on?
on shape or molecular configuration.
how does affinity chromatography work?
- antibodies are bound to resign beads
- as sample flows, antibodies will bind to antigenic epitope on protein of interest
a molecule of a given shape
or molecular configuration can be separated from other molecules
what is the key benefit of affinity chromatography?
Since antibody-antigen interactions are very specific, affinity chromatography can
be used to isolate and remove a single type of protein from a mixture of hundreds.
what is the challenge in affinity chromatography?
is finding a complementary molecule/antibody
to attach to the resin beads. Sometimes these already exist in nature. Sometimes these
can be made in the lab.
how does hydrophobic interaction chromatography separate protein molecules?
molecules using
the differences in their hydrophobicity in buffers of high salt concentration.
How does HIC (hydrophobic interaction chromatography) work?
resin beads are made of agarose with hydrophobic group.
proteins are mixed with ammonim sulfate or sodium sulfate (high salt buffer)
high salt buffer results in the protein to change shape and expose hydrophobic regions which will bind to the hydrophobic resin beads
the proteins with low hydrophobic-ness with elute first and then the ones with high hydrophobicness with elute lass
What is an open column chromatography
is gravity flow chromatography
What is open column chromatography for
small samples and small column volumes
What is the disadvantage of open-column chromatography?
buffer runs rather slowly & the quantitation is poor & the separation is poor
What is the advantage of open column chromatography?
gives the technician an idea of how to scale-up the purification process to larger, more effective columns.
How is a fast performance liquid chromatography different from open column chromatography?
FPLC- uses a pump= more efficient than just gravity
How does an FPLC (fast protein liquid chromatography) work?
- Tubing carries the buffer from reservoirs
- Tubing is fed through a pump mechanism that pushes the sample through the column and resin at different rates
- computers and pumps set the flow rate
- as the buffer flows through the resin, the beads separate the molecules based on their characteristics
- fractions leave column through frit membrane
- pass through exit tubing, sample detector (UV spec) and chart recorder or computer and into the fraction collector
When is FPLC used?
very large volumes
What is the HPLC (high perfomance liquid chromatograpy) What are the features that makes it different from other chromatography?
- uses tiny microcolumns
- resin-containing columns are made of metal that can withstand very high
pressures. - minute resin beads, provide increased SA for better surface area
What is HPLC used for?
can study tiny amounts
of proteins, DNA, and RNA.
WHat determines how molecules will be separated in the resin?
type of resin and buffers
What factors affects the binding of sample to the resin beads?
flow rate & pressure
What is an example that ion-exchange chromatography is used for?
soften hard water- water treatment companies
What property of the resin beads matter in size-exclusion (gel filtration) columns?
the diameter of the channels in the resin beads
determines which molecules can be separated from others.
What characteristic of resin beads matters in ion-exchange chromatography?
charge of resins
What 3 types of buffer is used in gel-filtration columns?
buffers used to dissolve the sample so the buffer carries the sample down the column
equilibration buffer- sets the charges on beads and proteins in the sample.
elution buffer- knock a bound protein off charged beads- high salt concentration
give an example of a common equilibration buffer
sodium monophosphate
What’s buffer exchange?
exchange, where the buffering compounds
in the sample solution are removed, and new buffering compounds take their place.
What is used or buffer exchange?
dialysis ( to remove old buffer from a previous process that will interfere with the new process and replace with new buffer)
In gel filtration columns, what characteristic should the bed have?
the bed must be long enough for a given concentration of
molecules to be trapped in the bead channels and separated from the larger
molecules.
Resin bead volume and sample concentration relatonship in ion-exchange column
there must be enough charged beads to bond all of the desired protein.
Resin bed volume and sample concentration relationship in affinity column
there must
be enough beads with bonding groups to bind all of the desired protein.
Why are spectrophotometers hooked up to most FPLC or
HPLC units?
to quantity the amount of sample passing through
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?
100 mL
Difference between QC and QA Department
QC department handles product quality and testing during product development, well before a sample is close to marketing.
QA department usually deals with quality
objectives, such as how certain objectives are met and reported, both
internally and externally, especially as a product is closer to marketing.
stability assay
length of time the product remains active
potency assay
effect of dosage on drug activity
toxicology assay
quantities of the drug toxic to cells and organisms
multispecies pharmokinetic assy
behavior of protein in nonhuman test animals
enzyme activity assay
degree of protein activity
ELISA assay
presence and concentration of the protein
what protocols does clinical testing include
- types of people accepted into trial * schedule of trials
- procedures * medications
- dosages * study duration
What type of biotechnology product undergoes clinical
testing/clinical trials?
Usually products or drugs that are used on humans
Why would a product fail?
- 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.
What factors affect a company’s product sales
effectiveness of the marketing team
- pricing decisions made by the company
- degree of patent protection afforded a product
- use of alternative therapies or products for the product’s target population
- timing of FDA approval of competitive products
- rate of market penetration for competitive products.
Proprietary rights contract define
so, an employee agrees to keep secret the
R&D of the company’s products.
What is used of finding new applications for products?
can save thousands of dollars in R&D costs.
How does changing a product on a molecular level benefit the company?
Each product version still must complete rigorous testing, but the
R&D costs are substantially reduced once the safety has been proved for the first
application. This translates into important profits for the company.
define Chromatography
separation of molecules through a stationary phase
define elution
process of releasing molecules from column
define fractions
what you collect at regular intervals
define frit
membrane filter at the bottom of the chromatography column
why can’t a patient go completely on placebo?
they would get sick again if the testing drug doesn’t work; usually use another medication they’re already on and compare with them
define sonification
sound waves to bust the cells open
solid phase of paper chromatography
paper
solid phase of thin layer chromatography
silica on glass plate
solid phase of column chromatography
column beads
how long do clinical trials last for?
2-5 years
Phase 1 of clinical trials
small group of human subjects (50 healthy people w/o disease)- safety test
Phase 2 of clinical trials
several hundred patients- checking for safety, dosage, & efficacy
Phase 3 of clinical trials
several thousand patients– checking for safety, dosage & efficacy; compare to existing drugs if not already
Phase 4 of clinical trials
post-marketing – follow up with patients across a course of several years
Column length effect on chromatography
long column- pressure back up but helps separate things that are similar
column width impact on chromatography column
big fat column- proteins move faster
pore size characteristics
change pore size based on protein size
sodium dodecyl sulfate
detergent that dissolves cell membranes
What makes a product marketable
patent protection, safe, effective, FDA approval, $$$
define Investigational new drug application
informs FDA of specific structure, function, production and testing processes of a potential new drug
must submit to FDA before clinical trials
New drug application (NDA)
show clinical trial results
to FDA
Patent protection –
process of securing a patent to an idea or tech
When is chromatography used
after recovery when protein has to isolated from other molecules
When harvesting broth cultures, how are cells separated from the broth
centrifugation or filtration
In column chromatography, what accomplishes the separation of molecules in a mixture?
size, charge and affinity
Difference in upstream and downstream process in biomanufactuing
upstream= fermentation
downstream= purifying proteins
What is paper chromatography used for?
aa composition of protein
STEPS- of paper chromatography
- filter paper used in solid phase
- filter paper used in chromatographic chamber with solvent
- solvent goes up the paper via capillary action
What does the distance molecules travel depend on in paper chromatography?
size and solubility
What molecules travel the farthest in paper chromatography?
smallest, most soluble molecules
what is ninhydrin
causes a.a. to colorize
what is Rf
distance amino acid have travelled compared to solvent –> to figure out what amino acid it is bc a.a. has their own Rf
how do you choose the right solvent in paper chromatography?
soluble in water, nonpolar inorganic solvents
What molecules would need to a nonpolar, inorganic solvent
lipids, proteins and amino acids
what are some examples of nonpolar, inorganic solvents
acetone or petroleum ether
What is the function of thin layer chromatography?
separates small molecules like amino acids
what are the steps of thin-layer chromatography
- glassplate + silica gel= stationary phase
- sample @ 1 side and buffer @ other side–> carries solute with it
Why would you use thin layer chromatography over paper chromatography?
bc its faster than paper
what is PAGE used or?
visualized which proteins separated into fractions
How would you determine separation in PAGE?
comparing bands
What is UV, ELISA and indicators used for
check separation
How does gel filtration work?
Sorts through different sizes of molecules
- size exclusion resin beads have tiny channels in which smaller molecules go through= longer time
- larger molecules skip channels & go through column= faster
What will the early fractions of gel filtration chromatography contain?
larger molecules
How does dialysis tubing work?
does buffer exchanges
- there is a sample of undesireable or desirable proteins in the dialysis tube which is tied off
- bag is placed in a large volume of the desired buffer
- unwanted molecules diffuse out of the bag throgh small pores that keep the protein of interest in the bag
- buffer changes and several hours go by..
5.broth is exchanged for chromatographic buffer
