monoclonal antibodies cheng

Question 1

what is the general conformation of an antibody?

Answer 1

Y shape conformation
– Disulfide bonds and non-covalent
interactions
– Heavy polypeptide chains (H)
– Light polypeptide chains (L)

Question 2

what are the constant and variable domains in an antibody?

Answer 2

Constant domains:
– CH1, CH2, CH3, CL
* Variable domains:
– Complementarity-determining regions
(CDR)
– Antigen recognition and binding

Question 3

what is CDC?

Answer 3

complement cascade can be activated to kill tumour cells and recruit immune cells

Question 4

how do antibodies produce angiogenesis?

Answer 4

antibodies bind to angiogenic factors eg VEGF produced by tumours that promote blood vessel development, thereby inhibiting new vessel formation

Question 5

how do mab stimulate apoptosis?

Answer 5

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

Question 6

what is the purpose of ADCC in mab?

Answer 6

immune cell binds to Fc of tumour-bound antibody. immune cells directly destroy tumour and release cytokines to attack more immune cells

Question 7

how does aggregation and particle formation vary with ph?

Answer 7

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

Question 8

how does covalent instability vary with ph?

Answer 8

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

Question 9

what are the known dosage forms of mab?

Answer 9

• 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)

Question 10

what are the limits and benefits of concentrated formulations?

Answer 10

• 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

Question 11

what are common techniques to produce mab?

Answer 11

• Filtration
• Drying (e.g. Freeze drying)
• Crystallization/precipitation (e.g.
  microparticles)

Question 12

what is the purpose of liquid concentrate?

Answer 12

• Increased in inter-molecular proximity
• Increased in protein-protein interaction

Question 13

define liquid concentrtae

Answer 13

Max. amount of protein that remains in solution, following 30 min of centrifugation at 30,000 g in the presence of co-solute

Question 14

how does viscosity affect syringability and injectability

Answer 14

the viscosity of the solution may affect the stability as a force is applied when syringing

Question 15

what are general formulation approaches to mab?

Answer 15

pH adjustment, choice of buffer system and ionic strength
* Other excipients, e.g. SAA and stabiliser etc.
* Container choice and needle size etc.

Question 16

how do you process a liquid preparation?

Answer 16

• 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

Question 17

what is continuous diafultration?

Answer 17

Constant retentate volume and product concentration
* One Diavolume (DV) = volume of filtrate collected equals starting feed volume

Question 18

how do you measure the process efficiency?

Answer 18

Process time (T) = V/(J x A),
where A is Membrane Area; V
is volume of filtrate generated

Question 19

how do you measure the filtrate flux?

Answer 19

Filtrate Flux (Jf) = Qf/area,
where Qf is filtrate flow

Question 20

what causes product losses?

Answer 20

*Micro-cavitation
*Air/liquid interface
*High protein concentration
*Process Temperature

Question 21

what are the steps in lyophilised powders?

Answer 21

WFI+buffer salts
buffer+solubiliser +ph modifier
add the drug
protein/ peptide solution
add the WFI
aseptic filtratioin
lyophilisation
lypophilised cake

Question 22

what is the manufacturing process of lyophilisation?

Answer 22

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

Question 23

what are the different types of thermal profile?

Answer 23

glass transitions
eutectic melting
devitrification

Question 24

how do you optimise lyophilisatioin?

Answer 24

• Thermal properties of the formula
• Avoid product collapse or melt back
• Set below critical temperatures
• Adjust chamber pressure according to
  the temperature

Question 25

what formulation considerations do you have in a lyophilised formulation?

Answer 25

• Optimal amount of solid in formula
• Type & concentration of excipients

Question 26

what is a lyophilised formulation made up of?

Answer 26

1. Buffering agent
   e.g. citrate, histidine, Tris
2. Bulking agent
   * e.g. glycine , mannitol
   * Mechanical support of cake
3. Stabilisers
4. Tonicity modifiers

Question 27

what is the purpose of stabilisers?

Answer 27

• 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.

Question 28

what is the mechanism of cryoprotection?

Answer 28

1. Preferential interaction
   – Preferential hydration at
   protein/peptide surface
2. Increase viscosity of the
   product

Question 29

what is the mechanism of lyoprotection?

Answer 29

1. Water replacement hypothesis
   – Replace loss of H bonds
2. Forming amorphous glassy state
   – Glassy state is highly viscous and stable

Question 30

why is the choice of container in a lyophilised formulation important?

Answer 30

– Prevent adsorption to container or/and photo decompositio
– Type I borosilicate glass
– ~ 20 - 50% of filled volume

Question 31

why is the choice of stopper important in a lyophilised formulation?

Answer 31

– Prevent change in moisture content or permeation of air
– e.g. butyl or halobutyl rubber

Question 32

what would reconsitution medium of a lyophilised formulation depend on?

Answer 32

Volume, pH, temperature of media etc.

Question 33

how do you optomise the final formulation of lyophilised formulation?

Answer 33

. 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