Week 6 and 9 - Biologics Flashcards
What are biologics
Medicines grown + purified from bacteria or yeast
- produced from living cells
- Used for monoclonal antibodies (largest group), growth factors, vaccines
- delivered via injection or infusion
Diff. between biologics and small molecules:
- biologics have highly specific binding (to modify / block function to target)
- less frequet dosing with biologics
- biologics have longer t1/2 = drug circulates longer
- biologics MAIN ISSUE = IMMUNOGENIC effects
- can have allergic reaction, antibodies can aggregate + trigger immune response
Have Fab region = antigen binds here
Have Fc region (Y end) = recycling of antibody
What is the difference between biologics, biosimilars and biobetters
Biosimilars:
- a biological product similiar to a reference product
- have amino acid sequence similar to refernce
- have no clinically meaningful differences to the reference
- e.g. effectiveness, safety, potency, purity
Biobetters:
- improvement of an already existing biologic to make it better
- are less likely to fail clinical testing
Explain the process of bioprocessing / bioprocess of mAbs
mAbs are proteins (made from amino acids)
- Cells are modified to generate active substance
- Cells are then multiplied to target density + frozen
- Active substance is produced in a bioreactor (nutrient solution used promotes growth)
- API is separated from cells + purified (using chromatography)
- drug is filled into vials in a highly sterile environment
- stored as frozen liquid, liquid in syringes (fridge)
Summary:
1. Cells are added to bioreactors containing nutrient media / solution (promotes growth / multiplication)
2. Separate the API from the by-products / other products using chromatography (purification step)
3. Purified API is stored in vials and as a frozen liquid or liquid (in sterile conditions)
Explain the formulation principles for specific biologics and biosimilars
Biologics are formulated as liquids or solids (for reconstitution) a.k.a. lyophilisation
- solids have longer shelf life
- Biologics are administered as injections or infusions e.g. IV, IM, SC
2 Types of Injections:
1. Pre-filled syringes (liquid form)
- max volume of 1 mL (measured dose)
- this form has better accuracy, convenience, patient compliance
- chemical degradation I.e. hydrolysis = less stable + has shorter shelf life compared to solid from
- physical degradation I.e. aggregation
- Dual chamber system (solid form)
- have lypholised drug near bottom and liquid near plunger (at top)
- when press plunger mix amount of liquid with solid = reconstitution
What testing are used for biologics and biosimilars
Stability Testing:
1. Long-term Testing:
- determien shelf life (time taken to lose 10% of drug)
2. Accelerated Testing:
- used to establish shlef life
- generate degradation profiles
3. Stress Yesting
- Reveals patterns of degradation
Explain some formulation issues with mAbs (specifcally liquid form)
- Prone to conformational changes (unfolding)
- chemical degradation or physical stress (e.g. shaking) can expose the hydrophobic a.acids
- when in water these a.acids try to avoid water contact = join other unfolded hydrophobic = aggregation
- aggregates can cause immune reaction in patients
Chemical degradation:
- hydrolysis
- oxidation
Physical degradation:
- extreme pH, High or low temp.
- physical stress (shaking)
- adsoprtion to surface of vial (can unfold protein or lwoer the dose)
How can we limit degradation
Lypholisation (solid form)
by FREEZING lypholisation (stored as solid for reconstitution with a liquid) USE CRYOPROTECTION
- this extends mABs shelf life + long term stability
- store lypholised med. in fridge to reduce aggregation
BUT freezing issues include:
- can get cold denaturation due to low temp.
- freezing can change pH, ionisation and solubility
- repeated freezing + thawing can cause aggregation (due to conformational changes)
- freezing forms crystals which leads to unfolding + breaking of mAB
What excipients are in protein and mAb formulations
- Buffer
- e.g. acetate, citrate, phosphate
- maintains required pH - Surfactants
- e.g. Polysoprbates (PS) 20 and 80 (tween 20 / tween 80)
- cover the intrerface of mAbs preventing unfolding
- PS degradation can contribute to aggregation, ensure they are sterile - Salt and tonicity modifiers
- e.g. NaCl
- IV injections require isotonic preparation
- IV, IM or SC require certain condition - Antioxidants
- e.g. EDTA (chelates metals)
- metals catalyse oxidation reaction - Protein stabilisers
- e.g. sugars (sucrose) or amino acids (arginine) - Lyophilistaion development
- e.g. PEG
Describe the structure of antibodies
Fab region - variable region
- where antigen binds (to antigen binding site)
- each antibody has 2 Fab regions
Fc region - fixed region (Y shape of antibody the ending)
- involved in recycling of antibody
How are antibodies recycled
Albumin is also recycled this way
- Fc region (on antibody) binds to Fc receptor (FcRn) on surface of cell
- Bound antibody + receptors is engulfed into cell and an endosome is formed
- As pH decreases mAb will move out of acidic endosome
- Unbound mAb will go through lysosomal degradation
- Bound mAb will move towards surface of cell, as pH increases the binding between mAb + Fc receptor breaks
- Antibody is released into circulation
What is ADA and its impact
ADA - Anti Drug Antibodies
ADAs are formed when immune system recognises there are foreign antibodies in body (i.e. mAb prescence)
- this triggers an immune response
- response is triggered by chimeric (-xumab) or partly humanised mAbs
what are the 2 types of ADAs
Neutralising ADA:
- binds to Fab region (on mAb) and limits its ability to bind to target antigen = ↓ efficacy of mAb treatment
Non-neutralising ADA:
- bind to Fc region of mAbs
- ↑ lysosoaml degradation + ↓ recycling of mAb
How has the evolution antibody synthesis changed over time
- Before antibodies was synthesised from mouse and human DNA = chimeric antibody
- would add human DNA to mouse antibody forming recombinant chimeric
- as antibody contains mouse DNA (in Fab region) when administered in body = higher chance of immunogenicity/ immune response due to it being recognised as foreign = ADA- suffix = -xumab
- Humanised antibodies = majority of antibody was from human DNA but still had small section of mouse DNA (in Fab region)
- Now have formation of fully humanised antibody = ONLY HUMAN DNA (no mouse DNA)
- less chance of immunogenicity
- have reduced immune reaction in patients
- suffix = -umab
Meanings:
-ximab = chimeric
-zumab = humanised
-umab = human
-o- = mouse
-a- = rat
What 4 MoA does mAbs have
- Block signalling pathways assoicated with GF receptors
- blocks ligand from binding - ADCC (Antibody Dependant Cellular Cytotoxicity)
- Antibody (Fab region) binds to antigen on target cell
- Fc region (of antibody) binds to natural killer cells = activation of NK cells - CDC (Complement Dependant Cytotoxicity)
- anyibody binds to antigen on cell + activates macrophages - ADCP (Antibody Dependnat Cellular Phagotycosis)
- Antibody (Fab) binds to antigen on cell surface
- Fc region binds to phagocyte = engulfs + degrades molecule = phagocytosis
What are the PK parameters (ADME) of mAbs
mAbs have non-linear PK
Absorption:
- when not adminstered via IV e.g. IM or SC = slow absorption due to bioavailability
- low molecular weight mAbs are absorbed well
Distribution:
- size + charge impact distribution (+ive charge = attach to cell surface)
mAbs get into tissue from blood via:
- diffusion / active diffusion
- paracellular (through space between cells)
- transcytosis (mAb binds to Fc receptor and is engulfed, transported through cell)
Elimination:
- Rate of elimination is influenced by dose (amount of mAb present) and expression of antigens (on cell surface)
- biologics have longer t1/2 than small molecule drugs (due to recycling)
- NO RENAL ELIMINATION (biologic size is too big, not filiteres)
- slow CL of mAb
