Ex 3 L3: Solutions for Injection Flashcards
Why solution formulation?
Solution formulations are popular for biologics because they:
-Are simplest and least expensive to manufacture
-Are convenient for patients and hospital personnel since they do not require reconstitution
-Can be inspected visually prior to admin
Clinical concerns for biologics in solution
Efficacy
Sterility
Side effects
-Dose-limiting immune response
Pain on injection (IM, SC)
-Volume administered
-pH (7.4)
-Tonicity, ionic strength
Concerns:
if it’ll work, if it is sterile, what side effects are present
Patients can form ADA - antidrug antibodies
-Over time, the body can start to say “this is not my protein, I do not like it, I need to make antibodies to protect myself from it”
-Every time drug is admin, anti-drug antibodies are produced, patient can no longer receive drug
Formulation concerns: For biologics in solution
All clinical concerns, plus:
Stability
-Aggregation
-Chemical stability (deamidation, oxidation)
-Shelf-life
-Storage conditions
Solution viscosity and ease of injection
-Can be affected by API concentration
Manufacturability
-Cost
-Manufacturing time
Formulation variables for biologics in solution
Solution properties:
-Ph
-Ionic strength, tonicity
-Drug concentration
-Volume
-Excipients (additives)
Container, closure
-Materials
Storage conditions
-Refrigerated vs room
-Frozen
Formulation variables: pH
Salmon calcitonin (sCT) is a peptide used to treat osteoporosis
-Rate of sCT degradation rate depends on pH
-pH of max stability is 3-4. Not a good pH for injection!!
-Additives make the stability worse.
Good pH for injection would be ~7.4
-Balancing what we need for patients with what the drug needs to be stable
Human calcitonin - not effective
Formulation Variables: Concentration
-Beta lactoglobulin is a milk protein, structure similar to Mabs
-Aggregate content increases with increasing protein concentration
-Since sc delivery requires small volumes, mAb concentrations are being pushed to 100mg/mL increasing aggregation risk
Higher concentration = greater aggregation
Turbidity is greater when aggregates are present
How do proteins aggregate
Bump into each other and stick
Proteins can partially unfold – expose hydrophobic domains
-Allows for proteins to stick together
Proteins can also unfold chemically
-Disulfide bonds
-Covalently linked
Three major types of aggregation:
-Colloidal interactions
-Chemical reactions
-Unfolding
More than one can happen at once
Most important process of aggregation
Unfolding
-partial unfolding is a death trap for stability
Protein folding
Protein folding – complex idea
Only recently been able to understand
How the primary sequence of a protein turns into fully folded structures
Total energy diagram (total state) starts with a lot of unfolded states at the top
-As energy gets lower, there are minima at “folding funnel”
-High entropy at the top, native state at the left
States we do not like
-Amyloid fibrils – problem in parkinsons (very low energy states)
-Amorphous aggregates that are lower energy than protein drugs
-Native state is what we want for activity
Aggregation and surfaces
Aggregation affected by surfaces
Start with protein in its native state
Greasy yellow bits – hydrophobic domains of the protein
In native state, protein wants them away from water
Hydrophilic domains on outside
When protein is exposed to surface
-Partially unfold – surface is more hydrophobic than the protein
Exposes native state
-Can also expose itself and partially unfold when exposed to air/water interface
-Does not necessarily come back off and refold – can come back and interact with itself before refolding
Dimer (two molecules sticking together)
-Higher order aggregates (multiple sticking together)
-Shaking the solution makes this worse
Not only does it expose more molecules to the interface, but you also make more interface (have a lot more surface) (higher exposure, higher surface area)
Especially at the three-phase boundary
Formulation Variables: Excipients
-Excipients can be used to help stabilize the protein
-Excipients that are preferentially excluded from the protein surface promote interactions with water and stabilize native protein strucutre
-Excipients that bind to the protein can lead to denaturation
-Exception: protein binding to ligands can stabilize native structure
Surfactants like to stay away from proteins
-Allows protein to associate with itself
We often add preferential exclusion solvents/cosolvents to help the protein stay together (prevent unfolding)
Excipients that bind to protein: things like urea, are good denaturing agents – We do NOT like those
-Protein binding to ligands can stabilize structure
Clinical effects of formulation
Erythropoetin (EPO, Epoetin) is used to treat anemia in renal disease
Formation of anti-EPO antibodies reduces drug effect and that of any naturally occurring EPO that remains
-Taking the drug for an endogenous replacement for something you have that is really low = now you not only do not receive extra EPO, you are making none at all
Pure red cell aplasia (PRCA) can result: sudden onset, anemia, death
In Europe, increased incidence of PRCA has been associated w/a change in container closure (stopper)
-Replaced one product (stopper) with another one with different properties, surfactants, and different aggregates with the protein
Formation of EPO aggregates has been suggested
What if solution formulation doesn’t work?
Store at refrigerated temp
Freeze
Freeze-dry (lyophilize) or spray-dry to create a dried powder for reconstitution
Re-engineer the protein molecule
-Go back to make the molecule stable
Abandon drug candidate
Practical considerations: solution formulations of biologics
-Store at recommended temperature
-protect from light if recommended
-Avoid agitation
-Examine vial for particulates prior to administration
-Be aware of the potential for adverse immune responses
COVID mRNA vaccines
The COVID mRNA vaccines are Suspensions of lipid nanoparticles (LNP) in solution. The mRNA is inside the LNP (mRNA LNP)
-The mRNA is not active without the LNP. Therefore, some people consider the mRNA LNP the API
Nevertheless, the formulation is a suspension
Lyphophilized mRNA LNP formulations are under development
mRNA is not active if LNP is not there
-Cannot be delivered to the place where it needs to do its job without the LNP
