Potential Questions For Final

Question 1

What are the methods of transformation?

Answer 1

Heat shock
CaCl2 transformation
Lipofectin and similar molecules
Electroporation
Microinjection

Question 2

To make the recombinant plasmid permeable to DNA molecules which of the chemicals is added?

Answer 2

CaCl2

Question 3

Generally a plasmid vector contains how many elements?

Answer 3

3

Question 4

Which of the following enzymes is required for end to end joining of DNA?

Answer 4

DNA ligase

Question 5

Which of the following is responsible for making a DNA copy from RNA?

Answer 5

Reverse transcriptase

Question 6

Can two DNAs cut with different restriction enzymes join together to form a recombinant plasmid?

Answer 6

Yes, provided the two enzymes have the same reaction site

Question 7

What are the differences between small molecule drugs and biological drugs?

Answer 7

Biologicals:
Bigger, higher molecular weight
Complex
Many options for modification
Produced in living cell culture
Can’t be characterized completely
Unstable
Immunogenic

Question 8

What was the first biologic product?

Answer 8

Insulin

Question 9

When do you extract primary metabolites and secondary metabolites?

Answer 9

Primary = log phase
Secondary = late log phase and early stationary phase

Question 10

What are secondary metabolites?

Answer 10

Antibiotics and phenolics

Question 11

At what level does rifamycin B work at?

Answer 11

Transcription level

Question 12

What is hayflick’s phenomenon?

Answer 12

The number of passages decreases when cells are harvested from older individuals

Question 13

What do most cells require as a supplement to promote cellular multiplication?

Answer 13

5-10% serum

Question 14

Why choose CHO cells?

Answer 14

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

Question 15

What is atryn?

Answer 15

Anti blood clotting protein antithrombin made using goats
Approved in 2009 by FDA

Question 16

What was the first animal used to make drugs?

Answer 16

Goats

Question 17

What are the types of biologics?

Answer 17

Vaccines
Hormones
Blood products
Cytokines
Gene and cellular therapies
Growth factors
Monoclonal antibodies
Fusion proteins

Question 18

Where do sources of variation come from in biologics?

Answer 18

Same gene sequence
Different vector
Different expression
Different cell line
Different bioreactor conditions

Question 19

What happens during upstream in bioreactors?

Answer 19

Sterilization
Preparation of media
Gathering raw materials

Question 20

What happens in production of bioreactors?

Answer 20

There is free cells, immobilized cells and enzyme bioreactor

Question 21

What happens in the downstream of bioreactors?

Answer 21

Product recovery

Question 22

What is the most common type of bioreactor?

Answer 22

Submerged type - stir tank

Question 23

What are the parts of a stirred-tank?

Answer 23

Sparger
Baffles

Question 24

What are spargers used for?

Answer 24

Introducing air bubbles of optimal size to maintain homogeneity of media

Question 25

What is baffles?

Answer 25

The metal plate used to prevent vortexing and ensure homogeneity of the culture media

Question 26

Explain an airlift bioreactor?

Answer 26

There is an inner and outer chamber. The inner chamber is the gassed region and the cells are lifted with air.

Question 27

Pros of a micro-carrier.

Answer 27

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

Question 28

What are the modes of bioreactor operations?

Answer 28

Batch Continuous Fed-batch

Question 29

Which bioreactor operation does not have an inlet or outlet component?

Answer 29

Batch operation

Question 30

What is there in bacterial proteins?

Answer 30

Inclusion bodies

Question 31

Why are inclusion bodies good?

Answer 31

Can easily be recovered to yield proteins More resistant to proteolysis

Question 32

What will inclusion bodies dissolve in?

Answer 32

SDS Urea Guanidine hydrochloride

Question 33

How do you prevent proteases?

Answer 33

Add protease inhibitors

Question 34

What is glycosylation?

Answer 34

Post translational modification - attachment of a sugar molecule (oligosaccharide)

Question 35

What can glycosylation affect?

Answer 35

Pharmacological function Immunogenicity Solubility Stability Serum half life

Question 36

What are potential contaminants?

Answer 36

Host Product Process

Question 37

In ion exchange chromatography, if your protein has a net + charge, what kind of chromatography would you use?

Answer 37

Cation exchange chromatography

Question 38

What is the charge on a raisin in cation exchange chromatography?

Answer 38

Negative

Question 39

What will treating an extracted protein with an SDS do?

Answer 39

Put a negative charge all over the protein and unfold the protein

Question 40

What are the two ways to detect a protein?

Answer 40

ELISA or Immunofluorescent

Question 41

The primary antibody binds to the _____

Answer 41

Antigen

Question 42

The secondary antibody binds to the _____

Answer 42

Primary antibody

Question 43

Which antibody has the fluorescent in indirect detection?

Answer 43

Secondary antibody

Question 44

Which antibody has the fluorescent in direct detection?

Answer 44

Primary antibody

Question 45

What is the difference between ELISA and immunofluorescent?

Answer 45

ELISA uses and enzyme for detection and immunofluorescent uses fluorescents for detection

Question 46

What must stay the same in biosimilars?

Answer 46

Presentation Dose Administration mode

Question 47

What do biosimilars involve?

Answer 47

Reverse engineering

Question 48

What are the product related substances and impurities?

Answer 48

Primary structure Biological function Higher order structure

Question 49

What are the process related impurities?

Answer 49

Stability Receptor binding and immuno-chemical properties General properties and excipients

Question 50

Where is the effort for each; stand-alone product and biosimilar?

Answer 50

Stand-alone - goal is to determine clinical effect so in clinical trials Biosimilar - goal is to determine similarity so in functional and physiochemical characterization

Question 51

Where are sources of variability for biosimilars in cloning, protein expression and production?

Answer 51

Vector and gene sequence you use Which host you use Cell expansion - different cell line Different bioreactor conditions

Question 52

Where are sources of variability for biosimilars in protein purification and formulation?

Answer 52

Different operating conditions Kind of chromatography Which filter paper supplier Methods of characterization and stability Formulation

Question 53

Differences between biosimilars and generic drugs?

Answer 53

Biosimilars: Complex Almost impossible to fully characterize; similar but not identical to reference Immunogenic $100-200 million/molecule

Question 54

What are the barriers to protein drug delivery?

Answer 54

Blood brain barrier Intestinal epithelial barrier Capillary endothelial barrier Enzymatic barrier

Question 55

What is passive targeting?

Answer 55

The ‘natural’ disposition pattern of the carrier system is utilized for delivery

Question 56

What is active targeting?

Answer 56

The concept where attempts are made to change the device by using “homing principle” to select one particular tissue or cell type

Question 57

When to target drugs?

Answer 57

Drugs with high total clearance Increase in rate of elimination of free drug Response site with relatively small blood flow

Question 58

What does the fate of particulate carriers depend on?

Answer 58

Size Charge Surface hydrophobicity Presence of homing device on their surfaces

Question 59

Where should the target site be when using liposomes?

Answer 59

In the blood

Question 60

What is PEGylation?

Answer 60

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

Question 61

What can antibiotics be produced by?

Answer 61

Bacteria, viruses, synthetic, and actinomycetes (type of bacteria)

Question 62

What is the difference between polyclonal and monoclonal antibodies?

Answer 62

Polyclonal: Cheap to produce Mixed populations of antibodies May bind to different areas on target molecule Tolerant or small changes in protein structure

Question 63

What makes something a drug candidate for MRDFs?

Answer 63

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

Question 64

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?

Answer 64

The SR BID tablet because it is the most similar to their current formulation.