Pharmaceutical Biotechnology Flashcards
What are the methods of transformation that we discussed? (5)
Heat Shock
CaCl2 Transformation
Lipofection
Electroporation
Microinjection
How many elements does a plasmid vector usually contain
3
OriC
Selectable marker
Restriction site
What does rifamycin do?
Both inhibit transcription by binding prokaryotic but not eukaryotic RNA polymerases
What is a restriction in using bacterial expression systems?
Incapable of producing glycoproteins
What are primary cell lines?
When cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide and grow.
Shorter passage life
Not used for longterm experiments
What is a cell line?
If the cells in a cell strain undergo a transformation process (spontaneous or induced changes in growth properties) that makes them “immortal”
What are the common culture surfaces used for cell culturing?
serum,
collagen,
laminin,
gelatin,
poly-L-lysine
What are CHO cell lines?
Type of cell line that has been used for stronger expression units and has high productivity
Can grow serum free
What is Pronuclear microinjection?
- Introduces the transgene DNA at the earliest possible stage of development of the zygote
- DNA is injected directly into nucleus of egg or sperm
What is Attryn?
Anti-blood clotting protein which was produced from the mammary glands of transgenic goats
First drug made from genetically engineered animals approved by the FDA
What is Erythropoietin?
Treat anemia and increases RBC count
What is the process of protein purification and formulation?
Protein recovery
Purification via chromatography
Purified bulk
Protein characterization
Formulization
What are the three cultivaiton systems for culturing cells?
Free in suspension
Attached to microspheres
Immobilized states as a monolayer
Bioreactors may be?
Liquid or solid state
What is the role of an impeller in a bioreactor?
Stir the cells
What is a micro-carrier bioreactor?
Uses glass beads for cells to grow on
What are spargers used for in bioreactors?
Introduce air into stirred tank bioreactor
What is the most commonly used bioreactor?
Stirred bioreactor
What is the important function of baffles in a bioreactor?
Prevent vortexing
How often is a “batch” media/product harvest/culture cells changed?
1/1/1
How often is a Fed-Batch media/product harvest/culture -cells changed?
> 1/1/1
How often is a continuous media/product harvest/culture -cells changed?
> 1/>1/1
What are the advantages of inclusion bodies?
Resistant to proteolysis and are protected from cleavage
What are the disadvantages of inclusion bodies?
Protein bound in inclusion bodies are biologically inactive
Usually denature in SDS, Urea, Guanine Hydrochloride
Addition of mercaptoethanol or DTT will enhance the solubilization of these inclusion bodies
What is the steps in inclusion body breakdown?
Intracellular product, breakdown the cells and removal of cell debris
Extracellular product, recovery of cell culture medium by removal of cells using centrifugation or filtration
Proteins become much more ___ at high temperatures
susceptible
What can affect a proteins characteristic and function?
Solubility
Stability
Serum half life
Pharmacological function
Immunogenicity
Which cell culture media contains additional proteins?
FSC
Growth factors
hormones
What are the potential contaminants in serum?
Proteins
viruses
bacteria
prions
endotoxin
proteases
What most frequent source of virus introduction is?
animal serum
What are pyrogens?
endotoxins that are potentially hazardous substances and cannot be removed via simple sterile filtration
What is the most common method of achieving pure protein samples?
Chromatography
What are the phases of chromatography?
Stationary phase
Mobile Phase
What is stationary phase of chromatography?
Insoluble matrix with which components of the mixture interact with during separation
What is mobile phase of chromatography
Solution just moves through the column
How many times are proteins run through a chromatography column?
2-3 times
How does ion exchange chromatography work?
Basically where the substrate is tagged with an anion or cation
What are the components of parenteral formulations?
SABAPA
Solubility enhancers
Anti-adsorption and anti-aggregation agents
Buffer components
Anti-Oxidants
Preservatives
Active ingredients
What is often added to help insulin not be adsorbed through the skin
Albumin will cover the hydrophobic regions allowing a competition between albumin and insulin for the surfaces
What are commonly used antimicrobial agents that are preservatives?
Thimersol, phenylmercuric nitrate, alcohol
What is a biosimilar?
Copy of a commercially available biopharmaceutical
Biosimilarity for antibodies can only be established how?
Through active evaluation in clinical trials and experiments with the reference product
What are interchangeables?
Interchangeables can be prescribed without the interference of a health care professional
What is the average time to produce a biosimilar?
7-8 years compared to 8-10 years
What are some barriers to protein drug delivery?
Enzymes
Intestinal barrier
Capillary barrier
Blood brain barrier
How can we increase the permeability of the absorption barrier?
Additon of FA/Phospholipids
liposome delivery
Decrease peptidase activity at the site of absorption
What is iontophoresis?
electric pads on skin that allows the drug to follow from positive to negative. Gets caught in blood stream and moves
Pulsatile secretion
What are the types of targeting systems we can use
Passive targeting (Macrophages)
Active targeting
What is PEGlyation?
Prevents an immune response and increases the ability of a drug to be soluble in water?
What is a disadvantage of liposomes?
Poor access to targets outside of blood
High resistance
What are the controlled released systems for parenteral delivery?
open loop and closed loop
Continuous infusion pump vs biosensory pump combo
What is an antisense oligionucleotide?
Short DNA analogue that hybridizes with the complementary mRNA
What does hybridization lean to?
Inhibition of gene expression
What is Luxturna?
First DFA approved gene therapy for retinol dystrophy Very expensive
What are hybridomas?
These are cancer cells fused with antibody-forming cells to produce more antibodies
Which phase are primary metabolites produced?
Log phase
Which phase are secondary metabolites produced?
Late log phase and early stationary phase
What is considered low risk in the manufacturing process?
Change filter supplier, Move equipment within the same facility
What is considered moderate risk in the manufacturing process?
Move to new production facility
What is considered high risk in the manufacturing process?
Change cell culture media, New cell line or major formulation change
Biosimilars are not
Second generation or generic drugs
What is the MOA of rifamycin?
Inhibit chain elongation of the RNA strand.
What are bacteria not capable of producing?
glycoproteins
What is Hayflick’s phenomenon?
Describes how primary culture cells have a limited number of passages before the cells just simply stop producing
What year did the FDA approve atttryn?
2009
What are the three parts of a biological process? with respect to bioreactors
Production, Product recovery, Sterilization of raw materials/air
Most bioreactors used in industry are?
Submerged type or liquid
The function of the impeller?
Stir it up
The function of the baffle?
Get rid of em vortexes
Function of the Sparger
Bubbles bubbles
What are micro-carrier bioreactors commonly used for?
Culture of immobilized mammalian cels
What are the reducing agents to break disulfide bonds?
Mercaptoethanol
Dithiothreitol
How can solubility be improved for protein delivery?
Proper PH
Add Lysine or Arginine
Surfactants
Sugars and glucose
Albumin is both an anti
Adhesion and anti aggregation
What is a limitation of osmotically driven systems/
Fixed release rates which isnt always desired
Antisense oligionucleotides can affect
Inhibition of 5’ cap formation,
RNA splicing
Activation of RNASE H
Block ribosomal subunit
How expensive is therapeutic antibodies for the treatment of disease?
300+ billion
