Monoclonal Antibodies (mAbs) + chromatography Flashcards
Give a short description of what mAbs are
Large glycosylated proteins composed of several domains which recognize and bind to ‘antigens’ with high affinity
State some differences between polyclonal- and monoclonal antibodies
Polyclonal antibodies: They are not similar, are produced by injecting pure antigen into animal, antibodies have a range of different binding activities
Monoclonal antibodies: Single type of antibody specified for one thing, produced using single mammalian cell clone, cells are clones which are grown in a fermenter
What host is used to produce mAbs?
Mammalian cell culture:
CHO cells (Chinese Hamster Ovaries) are considered the best
What would be a suitable DSP for mAbs? Put these operations in chronological order: Remove virus filtration- Concentration by UF - Chromatography by affinity - Chromatography by CEX - Centrifuge - Microfiltration - Fermenter
Fermenter - Centrifuge - Microfiltration - [Concentration by UF] (not usually included) - Chromatography by affinity - Chromatography by CEX - Remove virus filtration
Which type of chromatography is discussed in the course?
Packed bed liquid chromatography using resin packed into cylindrical column and liquid mobile phase
What is chromatography and state two uses?
High resolution method for separating contaminants that are often quite similar to product of interest.
Used for:
Purification of the product of interest (proteins)
Removal of contaminants
When in the process is chromatography used?
Chromatography is used toward the end of the purification step due to high selectivity and it is also very expensive
State the two types of operation for chromatography?
Elution chromatography
Adsorption or on/off chromatography (bind and elute)
What is the most common type of chromatography?
Adsorption / on-off chromatography (bind and elute is most common)
Describe Affinity chromatography
Specific molecule attaches to solid resin with aim of only attracting specific chromatography product
Describe Ion-exchange chromatography
Anionic or cationic ligands are covalently attached to solid
Describe hydrophobic chromatography
Hydrophobic are covalently attached to solid resin
Scale-up of chromatography rules of thumb
- Maintain linear flowrate
- Scale up by increasing diameter only and keep bed height the same
- Load is proportional to volume of resin
State the Langmuir binding model
Q’ = (Qmax * c’)/(Kd + c’)
How can binding capacity for chromatography column be determined?
Constructing a breakthrough curve
