Biological Agents 2 Flashcards
(26 cards)
What is the process involved in Upstream processing ?
Culture of producer cells
Name the processes involved in Downstream processing ?
- Extraction of protein from source material
- Concentrate/Initial purification
- Chromatographic purification
- Final product formulation
- Drying of product
- Packaging, labelling & shipping
Name 2 pro’s of bacterial production/purification
1) Easy and cheap to grow
2) Large amount of products
Name a con of bacterial production/purification
- It is made in an Insoluble form known as INCLUSION BODIES
What does IMAC stand for ?
Immobilised metal ion Affinity Chromatagraphy
Name the 5 types of Chromatography and briefly describe each type
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)
Describe rpHPLC
- 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
During IEC, what is the target protein eluted with ?
Increasing gradient of salt (NaCl)
What is the function of a Peristaltic pump ?
Gentle squeezing action moves fluid through the flexible tubing
During GFC, the largest protein in the sample is totally excluded and eluted from the column first. TRUE or FALSE ?
TRUE
When does Adsorption take place during HIC ?
When salt conc is high
When does Elution take place during HIC ?
When salt conc is decreased
Why characterise and monitor after C purification ?
Characterise to demonstrate Identity and prove Purity and Activity
Monitor for batch to batch variations
What does SDS-PAGE stand for and what is used for ?
Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis
Used to prove Purity during Characterisation
What is SDS and how does it work ?
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.
2D Gel electrophoresis separates proteins via Isoelectric focussing. TRUE or FALSE ?
TRUE
Describe the structure of an Antibody
- 2 heavy and 2 light chains
- 6 CDR’s (form Ag binding sites)
- Fc and Fab regions
What does the CDR determine ?
Specificity and Affinity
Name 5 ways mAb’s can be therapeutically useful ?
1) Ligand blockade
2) Receptor blockade
3) Receptor downregulation
4) Receptor activation
4) Cell depletion (produces mAb cytotoxic effect)
Name 3 reasons mouse mAb’s create problems to humans ?
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
Name the 4 types of mAb’s
1) Murine antibody
2) Chimeric antibody (66% human sequence)
3) Humanised antibody (90% human sequence)
4) Human antibody (100% human sequence)
Describe the PHAGE DISPLAY TECHNOLOGY technique;
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)
Why does the PHAGE DISPLAY TECHNOLOGY technique work ?
The expressed protein is directly linked to the DNA sequence coding for it
The antibody Herceptin blocks HER2 receptor and can be used to treat Metastatic breast cancers. TRUE or FALSE ?
TRUE
