Biological Agents 2

Question 1

What is the process involved in Upstream processing ?

Answer 1

Culture of producer cells

Question 2

Name the processes involved in Downstream processing ?

Answer 2

• Extraction of protein from source material
• Concentrate/Initial purification
• Chromatographic purification
• Final product formulation
• Drying of product
• Packaging, labelling & shipping

Question 3

Name 2 pro’s of bacterial production/purification

Answer 3

1) Easy and cheap to grow

2) Large amount of products

Question 4

Name a con of bacterial production/purification

Answer 4

• It is made in an Insoluble form known as INCLUSION BODIES

Question 5

What does IMAC stand for ?

Answer 5

Immobilised metal ion Affinity Chromatagraphy

Question 6

Name the 5 types of Chromatography and briefly describe each type

Answer 6

1) Ion Exchange C (IEC) - Based on differences b/wn protein charges at a given pH
2) Gel Filtration/Size Exclusion C (GFC/SEC) - Based on differences in mass/shape of different proteins
3) Affinity C (Ligand based, Antibody based, IMAC) - Based on differences b/wn protein and a ligand,etc
4) Hydrophobic interaction C (HIC) - Based on differences in surface hydrophobicity of proteins
5) reverse phase, High Performance, Liquid C (rpHPLC)

Question 7

Describe rpHPLC

Answer 7

• Takes place under acidic conditions (around pH 2)
• Protein interacts with Hydrophobic stationary phase
• Protein is eluted w/ Increasing gradient of organic solvent
• This technique is also known as a Denaturing technique
• Mostly carried out w/ 0.1% TFA (trifluroacetic acid) & Acetonitrite as the gradient

Question 8

During IEC, what is the target protein eluted with ?

Answer 8

Increasing gradient of salt (NaCl)

Question 9

What is the function of a Peristaltic pump ?

Answer 9

Gentle squeezing action moves fluid through the flexible tubing

Question 10

During GFC, the largest protein in the sample is totally excluded and eluted from the column first. TRUE or FALSE ?

Answer 10

TRUE

Question 11

When does Adsorption take place during HIC ?

Answer 11

When salt conc is high

Question 12

When does Elution take place during HIC ?

Answer 12

When salt conc is decreased

Question 13

Why characterise and monitor after C purification ?

Answer 13

Characterise to demonstrate Identity and prove Purity and Activity

Monitor for batch to batch variations

Question 14

What does SDS-PAGE stand for and what is used for ?

Answer 14

Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis

Used to prove Purity during Characterisation

Question 15

What is SDS and how does it work ?

Answer 15

A powerful detergent with a hydrophobic tail and a negatively charged sulfate head.

When SDS binds to protein in the sample, they become negatively charged and separate based on size.
Smaller protein move faster towards the positively charged electrode.

Question 16

2D Gel electrophoresis separates proteins via Isoelectric focussing. TRUE or FALSE ?

Answer 16

TRUE

Question 17

Describe the structure of an Antibody

Answer 17

• 2 heavy and 2 light chains
• 6 CDR’s (form Ag binding sites)
• Fc and Fab regions

Question 18

What does the CDR determine ?

Answer 18

Specificity and Affinity

Question 19

Name 5 ways mAb’s can be therapeutically useful ?

Answer 19

1) Ligand blockade
2) Receptor blockade
3) Receptor downregulation
4) Receptor activation
4) Cell depletion (produces mAb cytotoxic effect)

Question 20

Name 3 reasons mouse mAb’s create problems to humans ?

Answer 20

Mouse mAb’s are;

1) Immunogenic
2) Do not activate effector functions
3) Exhibit a short half life

2&3 = due to human Fc receptor not being able to recognise muse Fc region

Question 21

Name the 4 types of mAb’s

Answer 21

1) Murine antibody
2) Chimeric antibody (66% human sequence)
3) Humanised antibody (90% human sequence)
4) Human antibody (100% human sequence)

Question 22

Describe the PHAGE DISPLAY TECHNOLOGY technique;

Answer 22

Antibody that binds to a specific protein is selected from a pool of human antibodies and manipulated in the lab; to achieve the highest level of specificity for that protein

example of protein = TNF (tumour necrosis factor)

Question 23

Why does the PHAGE DISPLAY TECHNOLOGY technique work ?

Answer 23

The expressed protein is directly linked to the DNA sequence coding for it

Question 24

The antibody Herceptin blocks HER2 receptor and can be used to treat Metastatic breast cancers. TRUE or FALSE ?

Answer 24

TRUE

Question 25

What is the process of mAb prodution ?

Answer 25

1) Isolation of Immune cells
2) (insert into) Antibody producing cells
3) Hybridomas screened for production of desired antibody
4) Antibody-producing hybridomas = cloned
5) mAbs = produced

Question 26

PEG (polyethylene glycol) mediated myeloma cells; can be fused w/Hybridoma before being screened for desired antibody. TRUE or FALSE ?

Answer 26

TRUE