Lorna (Biologics and biosimilars) Flashcards
Biologic drugs and biosimilars
- Large molecules made using living cells (usually by genetically modifying cells.
- They are big and very complex molecules, often 200 to 1000 times the size of more common small molecule drugs.
- Examples are insulin and vaccines
FDA definition: “blood derived products, vaccines, in vivo diagnostic allergenic products, immunoglobulin products, products containing cells or microorganisms, and most protein products applicable to the prevention, treatment, or cure of a disease or condition of human beings” - Biosimilars are less costly imitations of biologics but they are different from generics in that they are not exact copies- e.g. neurofen and own brand
Biologic products
- There is a wide range of products such as vaccines, blood and blood components, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins
- Can be composed of sugars, proteins, or nucleic acids or complex combinations of these. They may also be living entities such as cells or tissues
- Isolated from a variety of natural sources- human, animal, or microorganism- and may be produced by biotechnology methods and other technologies
Products:
- Monoclonal antibodies
- Proteins intended for therapeutic use, including cytokines, enzymes
- Immunomodulators (non-vaccine and non-allergenic products intended to treat disease by inhibiting or modifying a pre-existing immune response)
- Cellular products, including products composed of human, bacterial, or animal cells
- Gene therapy products including nucleic acids, viruses, or genetically engineered microorganisms that mediate their effect by transcription or translation
- Vaccines
- Allergenic extracts used for the diagnosis and treatment of allergenic diseases and allergen patch tests
- Antitoxins, antivenins, and vemons
- Blood, blood components, plasma derived products
What do biologics have to offer?
- Ability to bind with high specificity (and highly selective to their targets) and affinity to a wide variety of molecules
- Dramatically improved patient outcomes e.g. in rheumatoid arthritis and cancer
(Humans are biological so work better with biologics).
Differences between biologics and ‘conventional’ drugs
Small drug molecules
- Chemically synthesised
- Low molecular weight
- Structure is known, easily identifies or characterised
- Less sensitive to environment
- Absorption across biological membranes is generally satisfactory
- Not necessary to use aseptic principles from initial manufacturing steps
- Variable specificity, may not be very high
Biological products
- Biotechnology products
- High molecular weight
- Structure is complex, not easily identified or characterised
- Very sensitive to environment (acid, heat, enzymatic degradation)
- Very poorly absorbed across biological membranes (have to be injected, too big to move across membrane, oral would be broken down by stomach)
- Susceptible to microbial contamination so necessary to use aseptic principles from initial manufacturing steps
- Target specificity very high (they are designed to bind to other biologics)
- Must be involved in more complex testing, Have to prove it works every time not just that it contains a certain atom (like in conventional drugs)
Examples of biologics
- Insulin
- Erythropoietin (EPO) (used for treatment of anaemia resulting from kidney disease)
- Human growth hormone (used for treatment of growth hormone deficiency)
- Pegfilgrastim (used to reduce the change of infection in people with certain cancers receiving chemotherapy)
- Etanercept (used in rheumatoid arthritis)
Categories of biologics
- Hormone- a substance produced by one tissue and conveyed by the bloodstream to another to effect physiological activity, such as growth or metabolism
- Interferons- proteins that are normally produced by cells in response to viral infection and other stimuli
- Interleukins- a large group of cytokine proteins. Most are involved in directing other immune cells to divide and differentiate
- Growth factor- a substance such as vitamin B12 or an interleukin that promoted growth, especially cellular growth
- Monoclonal antibodies- a single species of immunoglobulin molecules produced by culturing a single clone of a hybridoma cell. MAbs recognise only one chemical structure.
- Polypeptides- peptides containing ten or more amino acids
- Proteins- naturally occurring and synthetic polypeptides having molecular weights greater than about 10,000
- Vaccine- an agent containing antigens produced from killed, attenuated, or live pathogenic microorganisms, synthetic peptides, or by recombinant organisms. Used for stimulating the immune system of the recipient to produce specific antibodies providing active immunity and/or passive immunity in the progeny.
Antibodies
Antibody- immune system related proteins (immunoglobulins)
- Large Y-shaped proteins
- Two pairs of polypeptide chains, each pair containing a heavy and a light chain of different sizes
- Produced by the B lymphocyte and recruited by the immune system to identify and neutralise foreign objects, e.g. bacteria and viruses
- The binding activity of IgG molecules is generated by the variable domains of the heavy and light chains (the variable region has 3 complementarity determining regions (CDRs) and these 3 loops change, not the whole beta sheet)
Monoclonal antibodies (MAbs)
- Monoclonal antibodies are antibodies which have been artificially produced against a specific antigen.
- Extremely specific binding to target antigens
Monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell
MAbs mechanism of action
Function by three principal modes of action
1) block the action of specific molecules
2) target specific cells, or
3) function as signalling molecules
Blocking activity of therapeutic MAbs
Achieved by preventing growth factors, cytokines, or other soluble mediators reaching their target receptors. This can be accomplished either by the antibody binding to the factor itself or to its receptor.
e.g. Infliximab, Etanercept, and Adalimumab all block TNFalpha, and are indicated for a number of inflammatory conditions including rheumatoid arthritis, psoriasis, and Crohn’s disease
Targeting action of therapeutic MAbs
Involves directing antibodies towards specific populations of cells and is a versatile approach
Antibodies can be engineered to carry effector moieties such as drugs, enzymes, toxins, radionuclides, cytokines, or even DNA molecules to the target cells, where the attached moiety can then exert its effect.
e.g. antibody-drug conjugates
Function as signalling molecules
The natural effector functions of antibodies are associated with binding to Fc receptors or binding to complement proteins and inducing antibody-dependant cellar toxicity or complement dependant cytotoxicity.
(basically- get antibody to bind to something you want to destroy and the binding will trigger the activation pathway)
Example biologics- antibody-drug conjugates
- Three component system consisting of a potent agent, a linker and a monoclonal antibody
- The antibody binds to specific markers (antigens or receptors) at the surface of the cancer cell
- The conjugates is then internalised by the cancer cell
- The linker is degraded and the active drug released
- Antibody-drug conjugates enable the delivery of the attached drug selectively to cells that display the antibody’s target on their surfaces (less side effects and toxicity)
- This selectivity and targeted delivery reduces the side effects of the attached drugs which increases the number of drugs we can use as the side effects won’t be experienced (cytotoxic drugs for cancer treatment)
Production of therapeutic MAbs
- Complex- involves many steps
- Involves animals and cell culture
- The mouse hybridoma technology was an important step in the development of antibody technology and paved the way for the emergence of therapeutic monoclonal antibodies
- The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumour from this ‘immortal’ cell line is called a hybridoma
Hybridoma technology
1- Induce the production of B lymphocyte producing the antibody of interest in an organism - usually done by injecting a ‘foreign’ protein in mice
2- Extract B cells from the spleen of the mouse and add to a culture of myeloma cells (cancer cells). This results in the formation of hybridomas- cells formed by the fusion of a B cells and a myeloma cell
3- Screen for the hybridomas
4- Screen for desired antibody production. Initial collection of B cells is heterogeneous i.e. they do not all produce the same antibody. Therefore, hybridoma population also does not produce a single antibody.
5- Once sure that a certain hybridoma is producing the right antibody, that hybridoma can be cultured indefinitely and monoclonal antibodies harvested
Problems with murine MAbs
- Initial treatments using monoclonal antibodies produced using the hybridoma technology were associated with problems
- During repeat administration to humans in clinical trials, it was observed that the half-life decreased and the products became less effective with each injection
- This is due to the immunogenicity of murine proteins in humans and the rapid development of a human anti-murine antibody (HAMA) response in patients
- HAMA response neutralised the effectiveness of the murine antibodies and resulted in their rapid clearance from the body
Overcoming HAMA response
The solution was to make recombinant antibodies using antibody engineering
Antibody engineering
- The first MAbs were murine molecules and were recognised as foreign when injected into patients leading to problems.
- Developments in molecular biology made it possible in the early 90s to clone the genes of IgG molecules and as a result the genes of MAbs of interest could be cloned in eukaryotic expression vectors
- In this way, recombinant versions of any MAb could be obtained from diverse cell lines in a reproducible fashion
- Initial major application of antibody engineering was the possibility to create chimeric antibodies
- Antibodies are well conserved through evolution so possible to create chimeras by fusing murine variable domains (responsible for the binding activity) with human constant domains leading to ‘chimeric antibodies’, which are 70% human and considerably less immunogenic in humans.
- further development of antibody engineering made it possible to decrease even further the murine part of MAbs, using an approach called complementary determining region grafting leading to ‘humanised’ antibodies, which are 85-90% human and are even less immunogenic than chimeric antibodies
Human Hybridoma Technology
(fully human MAbs)
An in vitro process based on the immortalisation of B-cells from infected patients (can isolate B-cells from bone marrow)
- The B-cells harvested can be immortalised by fusion with Epstein-Barr Virus (EBV) in the presence of an oligonucleotide
- Immortalisation can also be performed with a human myeloma cell line
- The hybridomas can then be screened for specific antibodies
Transgenic Mouse Technology
(fully human MAbs)
An in vivo process based on the immunisation of a transgenic mouse.
- The mouse has been genetically engineered and bred for the expression of human Ig’s.
- The B cells harvested after immunisation can be immortalised by fusion with a myeloma cell line
- The hybridomas can then be screened for specific antibodies
(Mouse hybridomas but producing human antibodies because the mouse produces human Ig’s.)
Not everyone has the same sequence so there is still some immunogenicity but only a small amount.
Chinese hamster ovary (CHO)
- CHO cells are currently the predominant host used to produce therapeutic proteins.
- The process for development of a stable line starts with expression vector construction and transfection.
- After being transfected with plasmin bearing the antibody of interest genes, cells are screened for antibody production.
Summary of biologics production
Producing biologic products is more complex than production of relatively small molecules such as aspirin or penicillin. It requires:
- Genetic engineering
- Cell line development
- Upstream processing
- Downstream processing
- Formulation
- Quality control
- Regulation and registration
Key challenges with biologics
- Production
- Price
- Administration
- Immunogenicity
- Formulation and stability
- Biosimilars
Biosimilars
A biosimilar product is a biological product that is approved based on a showing that it is highly similar to an FDA-approved biological product, known as a reference product, and has no clinically meaningful differences in terms of safety and effectiveness from the reference product.
Only minor differences in clinically inactive components are allowed in biosimilar products.
Biosimilars are marketed after the patent for the branded product has expired.
Although they might be regarded as ‘generic’ medicines, they are not chemically identical, so the process for approval of licenses for marketing is not the same as it is for generic drugs.
