Biologics 1 Flashcards
What about biologics makes them more difficult to manufacture?
Complexity and size
Larger size means more problems in processing and stability of the product
Is it faster to get a biologic or small molecule drug to market?
Biologic due to several international regulations
Biologics can be used to…
Replace diseased tissue functionality e.g. protein hormones, blood factors, gene therapy and tissue engineering
Biologics are ______ than small molecules.
More versatile, can replace diseased tissue as well as modify pathways
More specific, small molecules have unspecific binding which can cause toxicity
Have more appropriate durations of actions and blood levels
Similar structures regardless of indication
More immunogenic
What is a shared problem of both biologics and small molecules?
Both can inappropriately target the wrong molecules
What are some of the different types of biologics on the market? (7)
Peptides e.g. teicoplanin Protein fragments mAbs e.g. Herceptin ADC's Viruses Vaccines New modalities such as LNP (lipid nanoparticles)
Define ‘bioprocess’
Specific process that uses complete living cells or their components
What are upstream and downstream processes?
Upstream - initial steps which start with mL quantities of mammalian cells engineered to produce a specific protein molecule
Downstream - following generation of Ab, isolation and purification must now take place
What are the 7 steps of antibody production?
1) Immunisation and isolation of splenocytes
2) Prep of myeloma cells
3) Fusion
4) Clone screening and picking
5) Functional characterisation
6) Scale up and wean
7) Expansion
What are the 6 steps of antibody manufacture?
1) Cell culture harvest
2) Protein A chromatography
3) Viral inactivation
4) Polishing steps
5) Viral filtration
6) UF/DF
How did Ab production begin?
Started with mice as creating Hu hybridomas was difficult
What was the problem with using mice?
Immunogenic reactions and rapid clearance as murine product
Lack of Fc effector functions
Over the last 2 decades, there has been a move towards…
Recombinant engineering
What is a chimeric antibody?
Mouse variable region (Fv, antigen binding)
What is a humanised antibody?
Mouse antigen binding loops (CDR’s)
What is a fully humanised antibody?
Produced via mice
What is the function of the Fc region of an antibody?
By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen
Also binds to various cell receptors, such as Fc receptors and other immune molecules such as complement proteins, in doing so it mediates different physiological effects including cell lysis and degranulation
What is the function of the Fv region of an antibody?
Also referred to as the variable domain/region
Most important region for binding antigens (more specifically, variable loops are responsible for binding antigen)
These loops are referred to as complementary determining regions (CDR’s)
Do all mAb’s work the same?
All mAb’s have roughly the same shape but have different CDR’s and antigens which lead to differences in therapeutic functionality
What are the 5 different mechanisms of action by antibodies?
1) Complement dependent cytotoxicity (CDC)
2) Conjugates
3) Apoptosis induction
4) Receptor (or ligand) blockade
5) Antibody dependent cell-mediated cytotoxicity (ADCC)
Explain CDC.
mAb’s induce CDC. When they bind to a surface antigen on a target cell, the complement pathway is induced leading to formation of membrane attack complex and target cell lysis. A typical complement protein involved in this sort of reaction is C1Q.
Explain conjugates.
mAb can be conjugated to a toxin or cytotoxic molecule to increase cytotoxicity
Explain apoptosis induction.
mAb can induce apoptosis on binding
Explain receptor (or ligand) blockade.
mAb blocks signalling complexes on binding
Explain ADCC.
Mechanism of immune defence where an effector cell of the immune system lyses a target cell following binding to the mAb on the surface. An example is NK cells or phagocytes, these attach to the antibody using Fc receptors.
How do mAb features contribute to PK?
Antigen binding region - comprise the CDR which may be involved in targeted mediated disposition, charge/PI mediated clearance, off-target binding
Glycan receptor - can cause characterisation problems following production, glycan mediated clearance and tissue distribution, problem due to variability in glycan level on different IgG’s
FcRn (neonatal Fc receptor) - present on IgG and albumin, favours recycling for long half-life
How are mAb’s administered?
IV, SC, or IM injections
What form are mAb formulations in?
Always liquid
Absorption for SC is…
Variable (20-95%), facilitated by lymphatic system
Is the rate of absorption of mAbs fast or slow?
Slow, maximal plasma conc. reached 1-8 days following SC/IM injection
How are mAb’s eliminated?
Not clearly understood
Eliminated by proteolytic metabolism degradation of large molecules into smaller peptides and AA’s reusable for protein synthesis
Other mechanisms include target mediated clearance, non-specific phagocytosis and Fc gamma receptor mediated clearance
Explain tissue mediated clearance.
Involves interaction between mAb and its pharmacological target, and represents the primary route of Ab clearance
Explain Fc gamma receptor mediated clearance.
Once the therapeutic IgG binds to the target (via Fab), the antigen-bound IgG binds to Fc-gamma receptors on effector cells (via Fc)
The resulting immune complexes are then cleared from the body through recticulo-endothelial system (RES)
Explain non-specific phagocytosis.
Ingestion of liquid into cell by budding small vesicles from cell membrane
Define ‘pharmacodynamics’
Refers to the pharmacological effects elicited by a drug in the body
What are the PD of mAb’s?
Differs from small drugs
PK/PD relationships are unique to each mAb
PK are markedly influenced by the biology of the target antigen (target-mediated drug disposition, TMDD)
What is one of the most important features of mAb’s?
Recycling
How is mAb recycling mediated?
Neonatal Fc receptor (FcRn)/Brambell receptor mediates recycling of albumin and IgG
What other region can bind to the FcRn and alter interactions?
Fv region, but this is less common
Define ‘isoelectric point’
The pH at which a molecule carries no net electrical charge
What other complex can be recycled through the FcRn pathway and what does this result in?
Antibody and antigen complexes
Can result in an accumulation of bound antigens in circulation and an extension of the half-life of antigens
To effectively remove antigen from the body, it is desired that the bound antigens be degraded whilst Ab are recycled
Molecules with a longer half-life…
All bind to FcRn
Proteins with a smaller molecular weight…
Have a shorter half-life as they are filtered by glomerular filtration due to their size
All IgG’s, natural and recombinant, are…
Glycosylated
Is glycosylation necessary for a mAb’s long half-life?
Not required
However, for Fc fusion proteins, the shorter half-life of an Fc fusion protein vs. IgG has been attributed to lower binding affinity to FcRn, glycan mediated disposition and receptor/fusion partner disposition
Does pI affect uptake of mAb’s into cells?
mAb’s with lower pI’s (more acidic) have lower rate of uptake into cells
This is as a result of repulsion between negatively charged cell surface and the charge of the antibody
This leads to a decreased tissue uptake and blood clearance
How can charge variation arise in mAb’s?
From Ab manufacturing processes due to chemical or enzymatic degradation via oxidation, deamidation, isomerisation of fragmentation
Charge differences can impact on…
PK and PD
What is one of the main problems faced by mAb’s?
Immunogenicity
Admin of therapeutic mAb may result in the formation of anti-drug antibodies (ADA’s)
What is the impact of formation of ADA’s?
ADA’s bind to the mAb, form immune complexes impacting on PK and pD, safety and efficacy
ADA’s can be neutralising (bind to epitopes on mAb needed for biological activity) or non-neutralising (bind to epitopes not needed for activity)
Does the immunogenicity of mAb’s vary and why?
Varies across species due to the different human fraction based on the type of therapeutic mAb (murine, chimeric, humanised or fully human) and hence animals are not predictive of human immunogenicity
Immunogenicity reactions are…
Very difficult to predict and can vary from patient to patient
What are some of the consequences of immunogenicity reactions?
Can cause hypersensitivity responses such as anaphylaxis and infusion reactions
Accelerated clearance of drug resulting in decreased safety and efficacy
Are fully human Ab less immunogenic than humanised ones?
No evidence of this
Novel formats can be grouped into which categories? (5)
Antibody fragments
Fusion proteins (as a result of conjugation of different protein parts)
ADC’s
Bispecific
Multi-specific (possessing multiple antigen binding sites)
How are novel formats more complex than mAb’s?
Different antigen-binding domains in the same molecule
Different molecular domains linked through flexible linkers
Heterogeneous product through conjugation, synthesis, physical chemical attributes
Use parts of proteins which are very unstable
There is an overall decreases in what with novel formats?
Conformation stability and stability
Important for storage and concentration that can be achieved
What happens if a novel format is too small?
Will be filtered by glomerular filtration
<7nm, <70kDa will be filtered
How does albumin avoid filtration?
Repulsion, it is negatively charged and so is the cell surface
What is the use of small proteins which are filtered?
Use as biomarkers
What are antibody fragments?
Skip the Fc part of the mAb
For some diseases the longer half-life and/or cell killing mechanisms of mAb’s are not required
Most prominent part is Fab
Give an example of an antibody fragment.
E.g. scFv, made of VH domain flexibly linked to VL domain, flexibility makes them prone to aggregation
What are fusion proteins?
A major class of new proteins Comprise a protein, peptide or receptor domain fused to the Fc region of the mAb Fc portion typically contains hinge region along with the conserved N-glycosylation site in the CH2 domain
What are ADC?
mAb employed as a drug delivery agent with chemotherapeutic drugs, immunotoxins, radioisotopes or cytokines covalently attached
After entering the cell, the linker (cleavable linker in Lys or Cys residues) is broken releasing the cytotoxic payload
What are multifunctional antibodies?
Bispecific or multispecific Ab contain 2 or more variable domains with specific affinity to bind different antigens
Bispecific formats comprise IgG-like and Fab fragment-based construct
