monoclonal antibodies cheng Flashcards
what is the general conformation of an antibody?
Y shape conformation
– Disulfide bonds and non-covalent
interactions
– Heavy polypeptide chains (H)
– Light polypeptide chains (L)
what are the constant and variable domains in an antibody?
Constant domains:
– CH1, CH2, CH3, CL
* Variable domains:
– Complementarity-determining regions
(CDR)
– Antigen recognition and binding
what is CDC?
complement cascade can be activated to kill tumour cells and recruit immune cells
how do antibodies produce angiogenesis?
antibodies bind to angiogenic factors eg VEGF produced by tumours that promote blood vessel development, thereby inhibiting new vessel formation
how do mab stimulate apoptosis?
antibody binding to cell receptor triggers signalling events that can lead to cell death. Signalling can be intensified when antibodies on surface are cross-linked by immune cells
what is the purpose of ADCC in mab?
immune cell binds to Fc of tumour-bound antibody. immune cells directly destroy tumour and release cytokines to attack more immune cells
how does aggregation and particle formation vary with ph?
at a lower ph acidic-unfolding and clip-mediated aggregation occurs
medium ph reversible dimerization
at a high ph pi precipitation and disulfide-linked aggregation occurs
how does covalent instability vary with ph?
deamidation and acid related hydrolysis- low ph
medium - iso asp/ cyclic imide
high ph- deamidation and proteolysis
oxidation due to active oxygen species from 3-9
what are the known dosage forms of mab?
- Simple solution
– Concentrate for infusion (e.g. bevacizumab) - Powder for reconstitution
– Unconjugated (e.g. transtuzumab) or conjugated
form (bentuximab vedotin, Trastuzumab
Deruxtecan (Erhertu)) - Radioimmunoconjugate (e.g. 90Y-ibritumomab
Tiuxetan)
what are the limits and benefits of concentrated formulations?
- Greater dose flexibility
- Less storage space
- Reduced cost of transport
- Lower production cost as liquid (?)
- Small volume (via SC or IM route)
- Reconstituted as IV infusion
- Processibility e.g. High viscosity
- Loss of yield due to dead volume etc.
- Increased cost due to greater overfilled volume etc.
- Appearance issue e.g. Opalescence
what are common techniques to produce mab?
- Filtration
- Drying (e.g. Freeze drying)
- Crystallization/precipitation (e.g.
microparticles)
what is the purpose of liquid concentrate?
- Increased in inter-molecular proximity
- Increased in protein-protein interaction
define liquid concentrtae
Max. amount of protein that remains in solution, following 30 min of centrifugation at 30,000 g in the presence of co-solute
how does viscosity affect syringability and injectability
the viscosity of the solution may affect the stability as a force is applied when syringing
what are general formulation approaches to mab?
pH adjustment, choice of buffer system and ionic strength
* Other excipients, e.g. SAA and stabiliser etc.
* Container choice and needle size etc.
how do you process a liquid preparation?
- Tangential flow filtration (TFF) or cross flow
filtration - Pressure-driven separation technique
- Ultrafiltration membrane or hollow fiber
- Diafiltration
- A means to clarify, purify and concentrate
what is continuous diafultration?
Constant retentate volume and product concentration
* One Diavolume (DV) = volume of filtrate collected equals starting feed volume
how do you measure the process efficiency?
Process time (T) = V/(J x A),
where A is Membrane Area; V
is volume of filtrate generated
how do you measure the filtrate flux?
Filtrate Flux (Jf) = Qf/area,
where Qf is filtrate flow
what causes product losses?
*Micro-cavitation
*Air/liquid interface
*High protein concentration
*Process Temperature
what are the steps in lyophilised powders?
WFI+buffer salts
buffer+solubiliser +ph modifier
add the drug
protein/ peptide solution
add the WFI
aseptic filtratioin
lyophilisation
lypophilised cake
what is the manufacturing process of lyophilisation?
Lyophilisation stresses destabilise unprotected
protein/peptide drugs
1. Low temperature & freezing stresses
2. Drying stress
Need to protect protein/peptide drugs
1. Optimising lyophilisation cycle
– Chamber pressure
– Temperature (condenser, shelf, product, glass
transition, collapse temperatures)
2. Addition of appropriate excipients
what are the different types of thermal profile?
glass transitions
eutectic melting
devitrification
how do you optimise lyophilisatioin?
- Thermal properties of the formula
- Avoid product collapse or melt back
- Set below critical temperatures
- Adjust chamber pressure according to
the temperature
what formulation considerations do you have in a lyophilised formulation?
- Optimal amount of solid in formula
- Type & concentration of excipients
what is a lyophilised formulation made up of?
- Buffering agent
e.g. citrate, histidine, Tris - Bulking agent
* e.g. glycine , mannitol
* Mechanical support of cake - Stabilisers
- Tonicity modifiers
what is the purpose of stabilisers?
- Maintain structure
- Cryoprotectants protect against from freezing denaturation
- Lyoprotectants protect against from drying denaturation
- Sugars/polyols, e.g. sucrose and trehalose
- Polymers and proteins: e.g. dextran, PVA, HPMC, albumin
- Surfactants: e.g. Tween 80
- Others: non aqueous solvent (e.g. PEGs, DMSO), amino
acids, salts etc.
what is the mechanism of cryoprotection?
- Preferential interaction
– Preferential hydration at
protein/peptide surface - Increase viscosity of the
product
what is the mechanism of lyoprotection?
- Water replacement hypothesis
– Replace loss of H bonds - Forming amorphous glassy state
– Glassy state is highly viscous and stable
why is the choice of container in a lyophilised formulation important?
– Prevent adsorption to container or/and photo decompositio
– Type I borosilicate glass
– ~ 20 - 50% of filled volume
why is the choice of stopper important in a lyophilised formulation?
– Prevent change in moisture content or permeation of air
– e.g. butyl or halobutyl rubber
what would reconsitution medium of a lyophilised formulation depend on?
Volume, pH, temperature of media etc.
how do you optomise the final formulation of lyophilised formulation?
. Cake physical attribute
2. Protein content
3. Moisture content
* Typically < 2% due to presence of strongly bound water
* e.g. loss-on-drying, Karl Fischer titration, TGA, GC, NIR
4. Ability to rehydrate or reconstitute
* Reversibility upon lyophilisation
* Particulate formation
7. Protein/peptide bioactivity, e.g. cell culture, animal model etc.
8 . Stability tests
