1. biotechnology basics Flashcards
this is the use of tissue culture, living cells, or cellular enzymes to produce a defined product
biotechnology
most biotechnology based pharmaceuticals are _____ based
protein
Most biopharma medications are developed for these three conditions
- cancers
- rare disease
- neurological disorders
is DNA synthesized from a single-stranded RNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a specific protein in a cell that does not normally express that protein, or to sequence or quantify mRNA molecules using DNA based methods.
cDNA (complementary DNA)
this is a series of events that takes place in a cell as it grows & divides
cell cycle
an increase in cell mass
cell growth
this is DNA that forms a closed loop that has no ends and provides antibiotic resistance to bacteria. e.g. plasmids
circular DNA
create copies of a particular gene for downstream applications, such as sequencing, mutagenesis, genotyping or heterologous expression of a protein.
gene cloning
the process where the genomes DNA is copied in cells. occurs during cell division
DNA replication
a group of enzymes that break the phosphodiester bond present within the polynucleotide chain of a DNA molecule
endonuclease
the process by which the information encoded in a gene is turned into a function.
gene expressions
is the process of making an RNA copy of a gene’s DNA sequence.
gene transcription
a small circular DNA molecule found in bacteria and other microscopic organisms.
plasmid
the process in which cells make proteins
protein synthesis
what are the building blocks of genetic materials?
DNA/RNA -> nucleotides & nucleosides
what are the building blocks of peptides/proteins
amino acids
what are the building blocks of oligosaccharides
carbohydrate sugars
what are the 5 nucleotides
guanine, cytosine, adenine, thymine and uracil
_____ (primary/secondary/tertiary/quaternary) protein structure is sequence of a chain of amino acids
primary
___________ (primary/secondary/tertiary/quaternary) protein structure occurs when the sequence of amino acids are linked by hydrogen bonds
secondary
________ (primary/secondary/tertiary/quaternary) occurs when certain attractions are present between alpha helices and pleated sheets
tertiary_
______ (primary/secondary/tertiary/quaternary) is a protein consisting of more than one amino acid chain
quaternary
there are four groups of amino acids: Glu and Asp are a part of what group
acidic side chains
there are four groups of amino acids: Lys, Arg and His are a part of what group
basic side chains
there are four groups of amino acids: Asn, Gln, See, Thr, Tyr are a part of what group
uncharged polar side chains
there are four groups of amino acids: Ala, Val. Leu, Ile, Pro, Phe, Met, Trp, Gly, Cys are a part of what group
nonpolar side chains
_____ and ______ are terms used in pharmacy to define how much os the active drug is available for us
stability and degradation
true/false: unique protein drugs can retain 100% of the chemical composition of the biologically active protein yet be biologically inactive
true
what processes cause degradation of traditional drugs
chemical instability
- oxidation
- hydrolysis
- racemizaitopn
what are the two main processes that cause the degradation of protein drugs
- chemical instability
- physical instability
this is covalent bond modifications of proteins via bond formation or cleavage (e.g. deamination, hydrolysis, oxidation, racemization, beta-elimination
chemical instability
this type of chemical instability process affects Amino acids that contain an amide (e.g. asparagine - Asn or glutamine (Gln)
amide gets converted to an acid (OH)
deamination
this type of chemical instability process affects peptide bonds of amides, especially when Pro is next to Asp
hydrolysis/proteolysis
this type of chemical instability is a major degradation pathway of proteins both in solution and lyophilized formulations. occurs in the presence of atmospheric oxygen
oxidization
______ containing amino acids are common targets of oxidation. this is due to the attraction of the nucleus to the valence electrons of _____ is not strong therefore easier to give up electrons for oxidation (e.g. cysteine and methionine)
sulfur
true/false: unpaired cysteines can be oxidized to form cysteine disulfide
true
this type of chemical instability is a reduced a SH on one cysteine can undergo exchange with another disulphide bond
disulfide exchange
true/false: a change in disulfide bonding will dramatically change the protein structure `
true
why would disulphide exchange or oxidation of an unpaired cysteine create a problem for proteins?
because such modifications will create new protein structures
all amino acids, except for ____, in mammalian biology are the L-amino acids
Gly
this process is the conversion of L-amino acids to D,L mixtures of amino acids.
racemization
what are the two labile amino acids that undergo deamination and racemization?
Asn, Gln
what is a labile amino acid that undergoes proteolysis and racemization
Asp
what are the five amino acids that under go oxidation
Met, Cys, His, Top and Tyr
what are the three amino acids that undergo beta-elimination and racemization
Ser, Thr, Cys
what is a labile amino acid that undergoes disulfide exchange and oxidation
Cys
this type of instability results in changes in protein secondary, tertiary and quaternary structures. no chemical bond formation or cleavage. predominant in large proteins
physical instability
true/false: peptides of >20 amino acids will degrade only by chemical instability since they have a complex secondary and tertiary structure
false -> peptides of < 20 AA will degrade only by chemical instability since they DO NOT have complex secondary and tertiary structures
what are the four types of physical degradation
- denaturation
- adsorption to surfaces/interfaces
- aggregation
- precipitation
this type of physical degradation is known as the disruption of secondary and/or tertiary structures. may or may not be reversible. is caused by heat, extreme pH and chemicals. typically involves the unfolding of proteins
protein degradation
when a protein is degraded, is it water soluble or water insoluble?
water insoluble - because the hydrophobic components become exposed when the protein is denatured
this type of physical degradation is the preference of molecules to localize at an interface (e.g. solid-liquid, air-liquid)
adsorption
proteins are ______ molecules which have both hydrophilic and hydrophobic amino acid residues
amphiphilic
true/false: larger proteins tend to adhere to surfaces more than smaller proteins
true
disulfide linked proteins are ___ (more/less) likely to unfold and will be less surface active
less
proteins may desorb, which is the removal of the protein from the surface and return it to the solution. once the protein desorbs, its hydrophobic residues/domains will be exposed to the water which will lead to either ________ or ________
aggregation or precipitation
________ results from the interactions between the hydrophobic portions of DIFFERENT denatured protein molecules
aggregation
immunogenicity is a complex immunological phenomenon affected by various factors. __________ have the potential to trigger an immune response in a patient, which can:
- decrease a drugs effectiveness
- cause harmful side effects
protein aggregates (or denatured proteins)
what are some additives used for prevent protein adsorption?
surfactants, other proteins (e.g. albumin), phospholipids
true/false: surfactants, proteins (e.g. albumin) and phospholipids are all amphillic and/or have surface activity
true
why is albumin used to prevent/minimize other protein adsorption?
- it is very water soluble because it is highly charged
- contains several disulphide bonds (very stable)
- competitor for inter-surface phenomenon (decreased chances of intermolecular interactions of the protein interested)
true/false: protein solutions should be shaken
false!!! air bubbles provide a new interface for proteins to adhere to
list some agents that are used to prevent protein aggregation
- sugars and salts (favour a compact state of the protein)
- polyols, PEGs and other polymers
- free amino acids (arginine)
_______ may aggregate/precipitate in the reservoir of _____ pumps; precipitates may block/obstruct the intake of the drug and therefore the patient will not receive the correct dose of ______
insulin
what are some reasons aggregation of insulin pumps may occur?
- body temperature
- agitation of insulin in reservoir with air
- adsorption of insulin to hydrophobic surfaces of air and PVC
true/false: total protein content (by protein assay) may not reflect its biological activity as its conformation can not be revealed by its protein contents. therefore, must perform a bioassay as well in addition to assaying for total protein
true
true/false: both chemical and physical degradation result in a loss go biological activity
true
k is known as the first order rate constant; the _______ (smaller/larger) the magnitude of k, the faster the degradation of active drug
larger
this type of degradation (physical or chemical) is assayed by a measure of the amount of intact protein using electrophoresis
chemical degradation
this type of degradation (physical or chemical) is harder to measure; measure the biological activity of protein (e.g. clot lysis assay to t-PA)
physical degradation
this drug is a tissue plasminogen activator (rt-PA). its indications include acute M.I. and pulmonary embolism
Activase (altepase)
true/false: degradation is additive
true
are most protein based drugs in the lyophilized form or solution form
most in lyophilized form b/c degradation reaction is accelerated when proteins are in solution
what are some methods used to enhance protein stability
- pH (buffer)
- decrease temperature
- light
- oxygen content (nitrogen flushing)
- excipients (antioxidants, chelating agents, ionic strength)
- lyophilization
- genetic engineering
- replace certain chemical reactive amino acids with those that are less reactive
- replace/intorduce certain amino acids to stabilize protein conformations
- biological activity must be retained
enhancement of in vivo protein stability
what are some ways that amino acids can be used for mutagenesis
- replace asparagine (undergoes deamination) with serine (avoids deamination)
- introduce additional Cys to enhance disulphide bridges and stabilize conformation
- introduce amino acids that improve hydrophobic packing to maximize hydrogen bonding
- introduce salt bridges
- replace Glycerol & Pro by Ala to reduce the conformational freedom of Gly and Pro
these two amino acids are alpha helical breakers
proline and glycine
this amino acid tends to break or kink helices because
- it cannot donate an amide hydrogen bond (having no amide hydrogen)
- its side chain interferes sterically
- its ring structure also restricts its backbone
proline
this amino acid tends to disrupt alpha helices because
- its high conformational flexibility (due to its small size) makes it entropically expensive to adopt the relatively constrained alpha helical structure
glycine
what are some challenges with protein drug delivery?
- poor oral bioavailability
- protein denaturation in the digestive system
- acid hydrolysis in the stomach
- enzymatic degradation
- poor absorption due to size
- poor absorption due to polar charge/distribution
what are some different example of parenteral delivery?
- IV administration
- subcutaneous injection
- continuous subcutaneous infusion (pumps)
- continuous intraperitoneal infusion
- intrathecal
what are some alternative formulations used for parenteral routes of protein drugs (alternative to solutions)
- liposomes
- micro encapsulations coated with polymers
what are some alternative routes of delivery of protein drugs besides injection
- inhaler
- intraocular
- intranasal
is an injection or continuous infection of protein drugs more beneficial
continuous infusion because it doesn’t spike out of therapeutic range and cause side effects like an injection would and does waste drug
what are the four types of liposomes
- conventional
- stealth
- targeted
- cationic
what are some advantages and disadvantages of intra-ocular drug delivery
ADVANTAGES
- convenient
- rapid systemic absorption
- bypass first pass metabolism
- dosing controlled precisely
DISADVANTAGES
- low systemic bioavailability
what are some advantages of nasal delivery of protein based drugs
- highly vascularized (quick absorption)
- milder pH (limited metabolism/destruction of protein based drugs)
- avoidance of first pass metabolism
this nasal spray contains calcitonin
it was approved in 2104 to treat certain bone problems (such as Pagets disease, postmenopausal osteoporosis) and to reduce high blood levels of calcium
Miacalcin (Salmon Calcitonin) nasal spray
what are the FOUR ways calcitonin affects blood calcium levels
- inhibits calcium absorption by the intestines
- inhibits osteoclast activity in the bones
- inhibits phosphate reapportion by the kidney tubules
- inhibits tubular reabsorption box calcium, leading to increased rates of its loss in urine
true/false: most proteins are kept at high temperatures
false - most kept at cold temperatures
exception is Alteplase
most proteins may adhere to either plastic or glass containers. the effectiveness of the products could be reduced by 3-4 fold due to adherence. ________ is added to reduce adherence
human serum albumin (HSA)
this medication is used for the treatment of cystic fibrosis. it is packaged in protective foil pouches provided by manufacture. stored in original light protective container. light protective pouches for travellers can be obtained from the manufacturer by patients who are going to travel.
dornase alfa (PULMOZYME)
this medications lyophilized form is light sensitive. solution after reconstitution is not light sensitive
Alteplase
what are some travel/shipping requirement for protein drugs
- stored in insulated, cool containers
- ice packs in warmer climates
- be cautious to protect the protein from freezing in the sub-freezing weather
- do not place protein products in direct contact with ice
- avoid using dry-ice
- place protein products in an insulated package not in a cargo container
true/false: when preparing protein drugs, it is important to shake them well
false - swirl the container - do not shake
true/false: most patients using protein products are immune compromised
true
what is the general sterility policy for protein drugs after reconstitution in the hospital
72 hours
what is the general sterility policity for protein drugs reconstituted at home
a 7 day expiration date is used, provided the product is stable and kept in the refrigerator
this is not recommended as most proteins will adhere to the filter. inline filters for TPN patients: protein products should be infused below the filter
filtration
what are some education points for those who are using parenteral protein drugs at home?
- make sure they are capable to handle, reconstitute and inject
- clean area and has a storage facility
- the patient is rotating the injection site
describe the normal clotting process
stage 1: blood vessels are damaged and bleeding starts
stage 2: blood vessels contract to slow the flow of blood to the injured area
stage 3: platelets stick to, and spread on, the walls of damaged blood vessels. these spreading platelets release substances that activate other nearby platelets which clump at the site if injury to form a platelet plug
stage 4: the surface of these activated platelets then provides a site for blood clotting to occur. clotting proteins like factor VIII (8) and factor IX (9) circulating in the blood are activated on the surface of the platelets to form a mesh-like fibrin clot
what is the difference in clotting between a healthy individual and someone who has a bleeding disorder?
HEALTHY INDIVIDUAL
the bleeding starts, vessel’s constrict, platelet plug, fibrin clot formed
BLEEDING DISORDER
the bleeding starts, vessels constrict, incomplete platelet plug, incomplete formation of fibrin clot
