Potential Questions For Final Flashcards
What are the methods of transformation?
Heat shock
CaCl2 transformation
Lipofectin and similar molecules
Electroporation
Microinjection
To make the recombinant plasmid permeable to DNA molecules which of the chemicals is added?
CaCl2
Generally a plasmid vector contains how many elements?
3
Which of the following enzymes is required for end to end joining of DNA?
DNA ligase
Which of the following is responsible for making a DNA copy from RNA?
Reverse transcriptase
Can two DNAs cut with different restriction enzymes join together to form a recombinant plasmid?
Yes, provided the two enzymes have the same reaction site
What are the differences between small molecule drugs and biological drugs?
Biologicals:
Bigger, higher molecular weight
Complex
Many options for modification
Produced in living cell culture
Can’t be characterized completely
Unstable
Immunogenic
What was the first biologic product?
Insulin
When do you extract primary metabolites and secondary metabolites?
Primary = log phase
Secondary = late log phase and early stationary phase
What are secondary metabolites?
Antibiotics and phenolics
At what level does rifamycin B work at?
Transcription level
What is hayflick’s phenomenon?
The number of passages decreases when cells are harvested from older individuals
What do most cells require as a supplement to promote cellular multiplication?
5-10% serum
Why choose CHO cells?
Good safety profile
Produce proteins with complex bio active PTMs that are similar to those produced in humans
Can grow in suspension culture
Can grow in serum-free chemically defined media
Allow gene amplification
Stronger expression units
Highly tolerant to pH, exogenous level, pressure, or temp
What is atryn?
Anti blood clotting protein antithrombin made using goats
Approved in 2009 by FDA
What was the first animal used to make drugs?
Goats
What are the types of biologics?
Vaccines
Hormones
Blood products
Cytokines
Gene and cellular therapies
Growth factors
Monoclonal antibodies
Fusion proteins
Where do sources of variation come from in biologics?
Same gene sequence
Different vector
Different expression
Different cell line
Different bioreactor conditions
What happens during upstream in bioreactors?
Sterilization
Preparation of media
Gathering raw materials
What happens in production of bioreactors?
There is free cells, immobilized cells and enzyme bioreactor
What happens in the downstream of bioreactors?
Product recovery
What is the most common type of bioreactor?
Submerged type - stir tank
What are the parts of a stirred-tank?
Sparger
Baffles
What are spargers used for?
Introducing air bubbles of optimal size to maintain homogeneity of media
What is baffles?
The metal plate used to prevent vortexing and ensure homogeneity of the culture media
Explain an airlift bioreactor?
There is an inner and outer chamber.
The inner chamber is the gassed region and the cells are lifted with air.
Pros of a micro-carrier.
Can provide extremely high productivity within a compact size
Has been used widely for culture of immobilized mammalian cells
Use porous glass beads to provide a large surface area of cells
What are the modes of bioreactor operations?
Batch
Continuous
Fed-batch
Which bioreactor operation does not have an inlet or outlet component?
Batch operation
What is there in bacterial proteins?
Inclusion bodies
Why are inclusion bodies good?
Can easily be recovered to yield proteins
More resistant to proteolysis
What will inclusion bodies dissolve in?
SDS
Urea
Guanidine hydrochloride
How do you prevent proteases?
Add protease inhibitors
What is glycosylation?
Post translational modification - attachment of a sugar molecule (oligosaccharide)
What can glycosylation affect?
Pharmacological function
Immunogenicity
Solubility
Stability
Serum half life
What are potential contaminants?
Host
Product
Process
In ion exchange chromatography, if your protein has a net + charge, what kind of chromatography would you use?
Cation exchange chromatography
What is the charge on a raisin in cation exchange chromatography?
Negative
What will treating an extracted protein with an SDS do?
Put a negative charge all over the protein and unfold the protein
What are the two ways to detect a protein?
ELISA or Immunofluorescent
The primary antibody binds to the _____
Antigen
The secondary antibody binds to the _____
Primary antibody
Which antibody has the fluorescent in indirect detection?
Secondary antibody
Which antibody has the fluorescent in direct detection?
Primary antibody
What is the difference between ELISA and immunofluorescent?
ELISA uses and enzyme for detection and immunofluorescent uses fluorescents for detection
What must stay the same in biosimilars?
Presentation
Dose
Administration mode
What do biosimilars involve?
Reverse engineering
What are the product related substances and impurities?
Primary structure
Biological function
Higher order structure
What are the process related impurities?
Stability
Receptor binding and immuno-chemical properties
General properties and excipients
Where is the effort for each; stand-alone product and biosimilar?
Stand-alone - goal is to determine clinical effect so in clinical trials
Biosimilar - goal is to determine similarity so in functional and physiochemical characterization
Where are sources of variability for biosimilars in cloning, protein expression and production?
Vector and gene sequence you use
Which host you use
Cell expansion - different cell line
Different bioreactor conditions
Where are sources of variability for biosimilars in protein purification and formulation?
Different operating conditions
Kind of chromatography
Which filter paper supplier
Methods of characterization and stability
Formulation
Differences between biosimilars and generic drugs?
Biosimilars:
Complex
Almost impossible to fully characterize; similar but not identical to reference
Immunogenic
$100-200 million/molecule
What are the barriers to protein drug delivery?
Blood brain barrier
Intestinal epithelial barrier
Capillary endothelial barrier
Enzymatic barrier
What is passive targeting?
The ‘natural’ disposition pattern of the carrier system is utilized for delivery
What is active targeting?
The concept where attempts are made to change the device by using “homing principle” to select one particular tissue or cell type
When to target drugs?
Drugs with high total clearance
Increase in rate of elimination of free drug
Response site with relatively small blood flow
What does the fate of particulate carriers depend on?
Size
Charge
Surface hydrophobicity
Presence of homing device on their surfaces
Where should the target site be when using liposomes?
In the blood
What is PEGylation?
Wont get taken up by macrophages - preventing immune response
The drug will have greater solubility in water
Very easy to attach targeting molecules
Decrease accessibility for proteolytic enzymes and antibodies
What can antibiotics be produced by?
Bacteria, viruses, synthetic, and actinomycetes (type of bacteria)
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal:
Cheap to produce
Mixed populations of antibodies
May bind to different areas on target molecule
Tolerant or small changes in protein structure
What makes something a drug candidate for MRDFs?
Drugs with neither high or low water solubility(in the middle would be okay)
High partition coefficient
Must be stable inside the body for extended period of time
Absorption cant be too slow or too fast
Can’t have high level of protein binding in blood(complex)
Can’t have narrow therapeutic window
If someone is on an extended release OD tablet but the pharmacy is out of stock, should you give them a SR BID tablet or an IR TID tablet?
The SR BID tablet because it is the most similar to their current formulation.
