interview

Question 1

When do we harvest cells?

Answer 1

When cells reach desired density. They are then moved to the downstream process.

Question 2

Downstream process outline (3)

Answer 2

1) cell capture and disruption
2) intermediate purification
3) polishing
4) mixed with inert ingredients, packed, sent to market

Question 3

Cell capture and disruption

Answer 3

quick separation of product from cells in the bioreactor: removes majority of water, enzyme, etc

Question 4

Cell capture and disruption - intracellular

Answer 4

keep pellet,

disrupt cell to release product through: centrifugation, filtration

Question 5

Cell capture and disruption - extracellular

Answer 5

keep media, discard pellet (ultrafiltration, centrifugation)

Question 6

Intermediate purification

Answer 6

removal of bulk contaminants (viruses, endotoxins, etc)

Question 7

Polishing

Answer 7

elimination of trace contaminants and impurities. Ex: partially unfolded peptide chain. Purity from 98% to 100%

Question 8

Purification Techniques (7)

Answer 8

1) centrifugation,
2) SDS-PAGE,
3) BCA assay,
4) Spectrophotometer,
5) Western Blot,
6) Filtration (TFF)
7) Chromatography

Question 9

SDS-PAGE purpose

Answer 9

to isolate proteins by size

Question 10

How are proteins converted to the same shape? (3)

Answer 10

1) SDS detergent,
2) beta-mecarptoethanol
3) heating samples to 95 degrees celsius for 5 min

Question 11

(SDS) detergent

Answer 11

solubilizes proteins to linear shape and gives them many negative charges. It disrupts secondary, tertiary structures.

Question 12

(SDS) beta-mecarptoethanol

Answer 12

reduces disulfide bonds

Question 13

(SDS) why is a sample of protein boiled in SDS and beta-mecarptoethanol?

Answer 13

Heat shakes up molecules allowing SDS to bind to hydrophobic regions and complete denaturation.

Question 14

SDS-PAGE loading buffer ingredients (5)

Answer 14

1) Tris-HCl: gives appropriate pH and helps samples move faster through gel,
2) SDS detergent: denatures proteins
3) Glycerol: makes samples viscous and sink into wells,
4) Bromphenol Blue: tracking dye, helps visualization of bands on gel,
5) Coomassie Brilliant Blue: stains gel after it’s done

Question 15

What type of information can you get from SDS-PAGE?

Answer 15

1) size,
2) relative concentration,
3) protein standards give you an estimate

Question 16

SDS-PAGE outline (11)

Answer 16

1) prepare gel,
2) seal gel in place with agarose and check for leaks,
3) mix your protein samples and loading buffer,
4) heat all samples to 95 degrees celsius for 5 min,
5) load samples into wells,
6) turn on apparatus at 150 volts until bands move about halfway, then 200 volts until the end,
7) turn off apparatus,
8) stain gel with Coomassie Brilliant Blue,
9) place gel on shaker,
10) destain gel,
11) photograph gel.

Question 17

Western Blotting use (4)

Answer 17

1) after SDS-PAGE,
2) gives MW,
3) it’s specific to the target protein,
4) detects very small amount of protein.

Question 18

Western Blot outline (6)

Answer 18

1) separate proteins by SDS-PAGE,
2) semi-dry apparatus: transfer proteins from gel to nitrocellulose membrane (mirror image: effective transfer)
3) block nonspecific sites on membrane with blocking buffer (5% dry milk powder) so only the target protein will stick to membrane,
4) incubate with primary antibody, wash unbound antibodies,
5) incubate with secondary antibody, wash unbound antibodies,
6) enzyme attached to secondary antibody converts: soluble, colorless substrate to colored, insoluble product. colored bands appear where protein interacts with primary antibody,

Question 19

What is the conjugated enzyme used in western blot?

Answer 19

(AP) alkaline phosphatase or horseradish peroxidase

Question 20

Calcium competent cells

Answer 20

to increase prokaryotic cells ability to incorporate plasmid DNA so it can be transformed later. (heat shock)

Question 21

Bioreactor (definition)

Answer 21

an apparatus to grow organisms under controlled conditions. used to produce pharmaceuticals, vaccines, antibodies, etc

Question 22

Parameters to monitor bioreactor: (4)

Answer 22

1) pH,
2) temperature,
3) agitation,
4) oxygen concentration

Question 23

Parameters to monitor cell growth (

Answer 23

1) microscopic count,
2) growth curve,
3) optical density
4) spread plates,
5) gram stains,
6) fixed volumes were taken at certain intervals

Question 24

Pichia pastoris (3)

Answer 24

1) high density biomass,
2) secretes recombinant protein into media,
3) doesn’t secrete many endotoxins (easier purification)

Question 25

cGMP (5)

Answer 25

1) #1 goal of pharmaceutical companies,
2) the minimum regulations enforced by the FDA,
3) inspection at least every 2 years,
4) failure to follow: adulterated drug, can’t be sold,
5) not only for manufacturing but also to storage and handling of all components involved in the process.

Question 26

Upstream outline (4)

Answer 26

1) cell isolation and cultivation,
2) cell banking,
3) cell expansion,
4) final harvest.

Question 27

Why BioMarin?

Answer 27

1) chronic, degenerative diseases,
2) BioMarin targets diseases that lack effective therapies,
3) orphan diseases (many are children) like MPS VI

Question 28

Naglazyme

Answer 28

enzyme arylsulfatase B

Question 29

Arylsulfatase B

Answer 29

part of group of enzymes present in lysosomes of liver, pancreas, and kidneys of animals. It hydrolyzes sulfates in the body by breaking down GAGs, which are large sugar molecules. The buildup of GAGs interferes with normal function of proteins in the lysosomes creating inflammation and then cell death.

Question 30

MPS VI symptoms

Answer 30

clouded cornea, deafness, abnormal growth, short stature, stiff joints, etc.

Question 31

What is Enzyme Replacement Therapy (ERT)?

Answer 31

Enzymes are introduced to the body to compensate for some deficiency. It is delivered via intravenous infusion

Question 32

Subculturing

Answer 32

The removal of medium and transfer of cells from a previous culture into a fresh growth medium to enable propagation of the cell line.

Question 33

When to subculture

Answer 33

During log phase, before cells reach confluence.