Biologics 🧬 Flashcards
Biologics (mAbs) formulation
• Biologics are either formulated as a liquid formulation or lyophilised powder (solid) (to be reconstituted before
Use)
• Administration is either subcutaneous, or i.v.
• Issues often linked to frequency of administration
• Adverse effects
Solid form advantages & disadvantages
• Dose and injection volume adjustable
• Can be developed as multi use formulations
• dual chambers allow precise volume & weight of protein
• Can be more expensive to couple a solid form to a delivery device (e.g., dual chambers)
Liquid form advantage & disadvantages
• More convenient to end user (precise concentration already prepared by company)
• Better patient compliance
• Better accuracy
• Chemical degradation hydrolysis thus less stable, limit shelf life, manipulation, etc..
• Physical stability more difficult to control aggregation (e.g., exposure to final fill finish operations) (step at which it could occur) (agglomeration has proteins are big flexible molecules that can lead to find conformational changes) Major obstacle as can start early on
mAbs conformation
Large molecules prone to conformational changes > may expose the hydrophobic acids buried inside
For mAbs and other therapeutic proteins in solution, the hydrophobic AA (amino acid) should be buried in the protein core and the hydrophilic AA should be in the shell. (Ensures stability but stress can disturb this)
Majority of all charged amino acids are on the surface of the mAb > makes antibody more hydrophilic
Majority of non polar & hydrophobic on the inside
mAbs stability
• mAbs (and other proteins) are not colloids
>Not a uniform distribution of the same charge
>Using DLVO theory (PHAR10100) is indicative at best.
• Need to consider attractive and repulsive forces but also solvation (ability to be in solution & contact in water)
> And consequently pH, buffer, salt (concentration and type) and concentration of co-solutes (to limit / maximise repulsion between the monoclonal antibodies). Consider PH of solution as want to increase solubility. mAbs isoelectric ph is 8 need it to be lower to PH 5-6 eg (not lower as it would irritate)
• Stress may lead to unfolding & ultimately to aggregation. (Hydrophobic parts exposed & will try to evade the contact with water)
• Aggregation not desirable
What stress can affect monoclonal antibodies?
Chemical degradation
• Oxidation, deamidation, hydrolysis
• May lead to instability then aggregation
>Exposure hydrophobic regions
>Exposure of cysteine residues of formation of disulfide bridges
Physical destabilisation
• Extreme pH (during formulation bio process)
• Shear forces (eg syringe & thin needle pressure but doesn’t happen when time administer just in earlier stages & an example to illustrate)
• Air-water interfaces (hydrophobic parts wants to get into contact with air)
• Adsorption to solid surfaces (wants to avoid contact with water) (eg wall of syringe)
• Freezing drying
• High pH or temperature changes
Formulation of proteins & mAbs
Most don’t need as small volume & conc & don’t need them stable for a long time eg not for infusions. Water only needed to reconstitute these but if need for a longer period of time:
Excipients
• Buffers
>Acetate, citrate, histidine or phosphate
>Formulations pH range often 5-6.5 (IgG pl approx. 8)
> Buffer concentration kept low to adapt to physiological pH upon administration
• Salt and tonicity modifiers
>Colloidal stability (attractive & repulsive forces. Changing salt amount in solution modulates charge of molecule & repulsion forces)
>i.v. injection requires isotonic preparation
>i.m. or s.c. injections may be able to handle hypertonic or hypotonic conditions
> Common excipient is NaCI, ArgI or sugars such as sorbitol. NaCl favours agglomeration however compared to the others. Sugars ensures solution is isosmotic
• Surface active agents (surfactants)
> mAbs flexible molecules with hydrophobic and hydrophilic regions
> Unfolding leads to aggregation
> Surfactants cover interfaces (air/liquid and solid liquid) thus limit unfolding
> Polysorbates 20 and 80 most common
> however polysorbates prone to degradation may contribute to aggregation & limit shelf life
> PS can under chemical degradation (oxidation & hydrolysis) as well as enzymatic degradation
> surfactants at concentrations above cmc (critical micelle concentration ) formation of micelles demonstrates that all interfaces have been covered
• Antioxidants
> Oxidation reaction catalysed by metals (may leach from surface of syringe)
> Use of EDTA to chelate metals to control oxidation
> Reducing agents such as glutathione can reverse oxidation
• Protein stabilizers
> Stabilisers are preferentially excluded (outside of the sphere of hydration of mAbs) : Lower interaction with protein but not hydrophobic (they are hydrophilic) leads to higher concentration of co-solute in bulk than in the solvation shell of the protein: increase the delta G unfolding > helps stabilise & important for higher volumes or lipholisation as these molecules pump the water molecules around and making the mAb more condensed. Requires far more energy to unfold the monoclonal body.
> Sugars e.g., sucrose
> Amino acids such as Arg (ArgHCI or ArgGlu) these provide both the isosmotic (shown previously) & protein stabiliser roles
• Lyophilisation development
> Use of PEG, sucrose, trehalose
> Same mechanism as stabilisers: exclusion by steric hindrance and maintained upon freezing
( when u lyophilise u pump out the water molecules which increases the concentration and the risk of unfolding increases as the encounter of molecules becomes more likely)
• Caveats to use of sugars as lyoprotectants
> Disaccharides susceptible to hydrolysis at low pH
> Hydrolysis of sucrose to glucose and fructose at pHs
Freezing mAbs (eg in plane in freezer)
• Low temperature extends shelf life
• But cold denaturation which happens when freezing sample (the sides & bottom & top of vile is where the cold comes from, water freezes first and all the rest of the molecules / ions concentrate at the centre of the vile) = more & more contact between mAb molecules
• Damage > results in change of pH, ionisation, solubility or H-bond energies.
• Repeated freezing and thawing cause aggregation by pH and concentration changes and by provision of nucleation points at ice water interface. Need to thaw & gently shake whole vile to ensure right concentration & dose to patient.
• Cryoprotection by sugars, polyhydric alcohols, AAs, work by preferential exclusion, lower cold denaturation and stabilise sample
Lyophilisation
Removal of water then adding it again before giving to patient. Removal of water preserves.
Loss of monomers happens as aggregates are created
• Lyophilised protein formulations have a greater long-term stability.
• These do undergo reversible conformational changes during the different steps of lyophilisation with render them prone to aggregation (and similarly again when reconstituted).
• Reactions and denaturation continue when lyophilised.
• Refrigerate lyophilised medicines to reduce aggregation rates
• Hygroscopic - sealed to avoid water vapour absorption.
• Advantage: allows us to modify concentration
mAbs mechanism of action
-Signalling pathway blocking: eg cetuximab / pantimab
Target: Receptors like EGFR (Epidermal Growth Factor Receptor) on cancer cells.(not necessarily on cell surface eg in RA adalinmumab binding to TNF alpha which is a soluble growth factor which moves in fluid & prevents it from binding to its receptor)
Mechanism: Prevents ligand binding to receptors.
Inhibits downstream signaling pathways (PI3K/AKT, RAS/ERK).
Outcome: Blocks cancer cell survival, proliferation, and growth.
- Antibody-Dependent Cellular Cytotoxicity (ADCC): eg tafasitamab
Target: Antigens like CD19 on cancer cells.
Mechanism: Antibodies recruit NK cells by binding to Fc gamma RIII receptors.
NK cells release cytotoxic molecules (granzyme and perforin). FC gamma RII receptor of natural killer cell bind to FC region of antibody and induces lysis of cell.
Outcome: Cancer cells are lysed by NK cell-induced cytotoxicity.
-Complement-Dependent Cytotoxicity (CDC) : eg Naxitamab
Target: Antigens like GD2 on cancer cells.
Mechanism: Antibodies activate the complement system by recruiting C1q. (Compliment protein)
Induces formation of the Membrane Attack Complex (MAC).
Outcome: Cancer cells undergo lysis through complement-mediated action.
Antibody-Dependent Cellular Phagocytosis: (ADCP) eg tafasitamab
Target: Antigens like CD19 on cancer cells.
Mechanism: Similar to ADCC but now FC gamma receptor on macrophage recognises mAb FC receptor. Antibodies engage macrophages via FcγRI receptors. Macrophages engulf and digest cancer cells through phagocytosis.
Outcome: Cancer cells are destroyed by macrophages.
Antibodies & FcRn receptors
Antibodies bind to Fc receptors (specifically the neonatal Fc receptor, or FcRn) for recycling to extend their half-life and maintain their effectiveness in the body. Here’s why this process is crucial:
- Protection from Degradation
Endocytosis: Antibodies, like IgG, are taken into cells through a process called endocytosis. Once inside, they enter acidic endosomes.
Without FcRn binding, antibodies would be directed to lysosomes for degradation, leading to their breakdown and loss of function. - FcRn-Mediated Recycling
Binding in Acidic Environments: FcRn binds to the Fc region of antibodies in the acidic pH (around 6.0) of the endosome.
Release at Neutral pH: After binding, FcRn protects the antibody from degradation and transports it back to the cell surface, where the neutral pH (7.4) causes the antibody to be released into circulation. - Prolonged Circulation
Recycling allows antibodies to avoid degradation and return to the bloodstream, significantly extending their half-life.
IgG antibodies and albumin are examples of proteins whose longevity depends on FcRn recycling. - Efficient Use of Resources
Recycling reduces the need for constant production of new antibodies by the immune system.
It ensures that functional antibodies are reused, optimizing immune defense mechanisms. - Therapeutic Applications
Monoclonal Antibodies (mAbs): Engineers often optimize the Fc region of therapeutic antibodies to enhance binding to FcRn, improving their stability and half-life.
Drug Delivery: FcRn is used to extend the half-life of Fc-fusion proteins or antibody-drug conjugates.
In Simple Terms
Binding to Fc receptors like FcRn acts like a rescue system for antibodies. Instead of being destroyed inside cells, they are “rescued” and sent back into the bloodstream to continue their job of fighting pathogens. This process helps antibodies last longer and reduces the body’s burden of constantly making new ones.
Q: What is the signaling pathway blockade mechanism of mAbs?
mAbs bind to receptors or soluble ligands to block receptor activation and signaling.
Example: Adalimumab binds TNF-alpha, preventing receptor interaction.
Q: What is ADCC (Antibody-Dependent Cellular Cytotoxicity)?
Fc region of mAbs binds Fc gamma receptors on NK cells, triggering cell lysis.
Example: Tafasitamab targets CD19 receptors.
Q: What is CDC (Complement-Dependent Cytotoxicity)?
mAbs bind target receptors and recruit complement proteins (e.g., C1q).
Leads to membrane attack complex formation and cell lysis.
Q: What is ADCP (Antibody-Dependent Cellular Phagocytosis)?
Fc region of mAbs binds Fc gamma receptors on macrophages, triggering phagocytosis.
Q: What are the main routes of administration for mAbs?
IV, subcutaneous (SC), or intramuscular (IM).
SC and IM routes have slow absorption due to the large molecular size of mAbs.
Why do mAbs have restricted tissue penetration?
Large size (~165 kDa) limits diffusion; penetration relies on transcytosis or leaky vasculature.
Q: How are mAbs eliminated?
Proteolytic degradation in lysosomes after endocytosis.
No renal elimination due to large size (above glomerular filtration cutoff).
Q: What is FcRn recycling and its role in mAb PK?
FcRn binds mAbs at acidic pH in endosomes, protecting them from degradation.
At neutral pH, mAbs are released back into circulation, extending half-life.
Q: What is Target-Mediated Drug Disposition (TMDD)?
mAbs binding to antigens are internalized and degraded, influencing clearance.
High antigen levels increase mAb clearance.
Q: What are Anti-Drug Antibodies (ADAs) and their types?
ADAs are immune responses against mAbs. Most biological drug products elicit some level of anti-drug antibody (ADA) response. This is because biological components are detected by the body and induce an immune (humurol) response resulting in the formation of antibodies.
Neutralizing ADAs: Bind Fab region (where they’re supposed to bind to antigen receptor) , blocking antigen interaction. (The upper Y section )
Non-neutralizing ADAs: Bind Fc region, preventing FcRn recycling.
Q: What affects mAb half-life?
FcRn-mediated recycling extends half-life.
High antigen levels reduce half-life due to increased lysosomal degradation.
Q: How do mAbs differ from small molecules in distribution?
Small molecules diffuse easily; mAbs rely on transcytosis.
mAbs have smaller distribution volume limited to extracellular space.
Q: How are small molecules and mAbs cleared differently?
Small molecules are renally excreted; mAbs are cleared via lysosomal degradation.
