Lectures 19/20 Flashcards
Peptides, Proteins, and Biopharmaceuticals - Topp
biologic drugs
medicinal agents derived from living systems, in contrast to more conventional “small molecules” drugs produced by chemical syntehsis
types of biologic drugs
recombinant proteins
plasma immunoglobulins (IVIG)
some peptides
vaccines
DNA
RNA, mRNA
cell and gene therapies
cell-based therapies
AAV vectors
gene editing (CRISPR)
peptides and oligonucleotides (tides)
have properties intermediate between small and large molecules
also included in biologics
common features of biologics
biological origin. (animal or cellular/molecular biology)
high molecular weight
higher order structure
early biologics – 1920s
making insulin in the 1920s through extraction of porcine insulin from pig pancreas at Eli Lilly
high sequence homology among human, porcine, bovine insulin
early biologics – 1957
making flu vaccine in eggs
eli lilly workers in greenfield, IN
candidate vaccine viruses (CVVs) are grown in chicken eggs –> CVVs are injected into fertilized chicken eggs and incubated for several days –> fluid containing virus is harvested from the eggs –> vaccine viruses are inactivated (killed) –> virus antigen is purified
recombinant proteins in the 1980s
created the central dogma of biology (DNA to RNA to Protein)
first recombinant form of human insulin created by Eli Lilly in October 1982
bacterial cell as protein factory
development of methods to grow cells in culture, isolate, and purify the protein produce
recombinant proteins (mAbs)
leading recombinant protein drugs
first mAb, OKT3, approved in 1986 to prevent transplant rejection
high molecular weight
produced in mammalian cells because posttranslational glycosylation is important to their structure and function
CAR-T
approved in august 2017
chimeric antigen receptor (CAR) T-cel therapy
examples – Kymriah, CTL019, Tisagenlecleucel, Novartisc
contrast – allogeneic cell therapy
Kymriah
first living drug
autologous T-cell therapy
T-cells are removed from a patients blood –> transfected with an antigen receptor that recognizes a cancer antigen –> grown in culture –> return to the patient and infused
mRNA vaccines
new revolution, circa 2020
contains mRNA in lipid nanoparticles
example – Covid-19
mRNA codes for the spike protein of the virus
rapid introduction has started the revolution
LNP
contains four types of lipids that condense mRNA and enable delivery to the cytoplasm
characters of biologics
DNA
mRNA
cells
proteins
viral vector
animal source
biologics timeline
early biology – isolated from animal tissues
recombinant proteins – expressed in transfected cell lines; act as protein factories
role of cells in biologics
protein factory
therapeutic agent (ex. CAR-T)
therapeutic target
role of viral vectors in biologics
transfection reagent
therapeutic agent
therapeutic target (ex. COVID-19)
role of animal tissue in biologics
flu vaccines (chicken)
role of proteins in biologics
therapeutic agent
therapeutic target (receptor)
role of DNA in biologics
instructions for transfected cells
therapeutic agent (ex. DNA vaccines)
why is biologic dosing usually parenteral?
due to biologics being degrading extensively in the GI tract
exception - oral vaccines, some orally administered peptides
examples - solution for injection, pen or autoinjector, pre-filled syringe, lyophilized powder for reconstitution
why are solution formulations popular?
simplest and least expensive to manufacture
convenient for patients and hospital personnel since they do not require reconstitution
can be inspected visually prior to adminsitration
clinic concerns for biologics
efficacy
sterility
side effects (dose limiting immune response)
pain on injection (volume administered, pH, tonicity ionic strength)
formulation concerns
all clinical concerns and
stability
solution viscosity and ease of injection (can be affected by API concentration)
manufacturability (cost, manufacturing time)
factors of stability
aggregation
chemical stability (deamidation, oxidation)
shelf-life
storage conditions
formulation variables
solution properties (pH, ionic strength tonicity, drug concentration, volume and exicipients)
container, closure (materials)
storage conditions (fridge vs room vs frozen)
pH variable
formulation variables
salmon calcitonin (sCT) peptide is used to treat osteoprosis with the rate of degradation depending on pH
pH of maximum stability is 3-4; not a good pH for injection
additives make the stability worse
concentration formulation variables
example beta lactoglobulin
milk protein, structurally similar to mAbs
aggregate content increases with increasing protein concentration
since SQ delivery requires small volumes, mAb concentrations are being pushed to 100mg/mL, increasing aggregation risk
higher concentration = ? aggregation
greater aggregation thus greater turbidity
protein aggregation mechanisms
chemical reaction
colloidal interactions
unfolding (highest energy)
partial unfolding at air/liquid interface
is worse with agitation
especially prominent at three-phase boundary (container, air, solution formulation)
stabilized protein structure
excipients that are preferentially excluded from the protein surface
promote interactions with water and stabilize native protein structure
exception – protein binding to ligands can stabilize native structure
de-stabilized protein structure
cosolvent surrounds protein in preferential binding
can lead to denaturation
erythropoetin
EPO, Epoetin
used to treat anemia in renal disease
formulation of anti-EPO antibodies reduces drug effect and that of any naturally occurring EPO that remains
pure red cell aplasia (PRCA)
can result in sudden onset anemia, death
in europe, increased incidence of PRCA has been associated with a change in container closure (stopper)
what if the solution formulation doesn’t work?
store at refrigerated temperature
freeze
free-dry (lyophilize) or spray-dry to create a dried powder for reconstitution
re-engineer the protein molecule
abandon drug candidate
practical considerations for solution formulations of biologics
store at recommend temp
protect from light if recommended
avoid agitation (dont shake my baby!!!)
examine vial for particulates prior to adminstration
be aware of the potential for adverse immune responses
covid mRNA vaccine
suspensions of lipid nanoparticles (LNP) in solution which the mRNA being inside of it
mRNA is not active without LMP
what is prefilled syringes, pens, and autoinjectors?
medical devices
not formulations
the solution inside it is a formulation so together it is called a combination product
advantages of pre-filled syringes, pens, and autoinjectors
ease of use, convenience
easier to transport than vial and syringe
discrete
increased patient compliance
reduced risk of dosage error
reduced risk of product contamination
disadvantages of pre-filled syringes, pens, and autoinjectors
higher cost than vial plus syringe
cannot mix two drugs
drug waste due to priming
greater surface to volume ration, presence of lubricants, can induce aggregation of protein drugs
pre-filled syringe components
syringe barrel
luer lock
finger grip
plunger
gasket
top cap
location of leur lock
in between the top cap and the syringe barrel
components of insulin pens
pen cap
outer needle cap
inner needle cap
needle
protective seal
rubber seal
insulin reservoir
dose window
dosage knob
injection button
common features of pre-filled syringes, autoinjectors, and pens
drug solution
needle
piston/plunger
housing
special concerns for pre-filled syringes, autoinjectors, and pens
higher surface to volume ratio
lower total volume
syringe lubricants/oils
addition of syringe lubricants or oils
change interaction of proteins with solid surface (hydrophobic lubricant lines container)
droplets in solution (provide additional hydrophobic surface area)
advantages of lyophilized powders
reduced rates of chemical and physical degradation
improved drug stability and longer shelf-life
refrigerated storage is not usually needed
can use lyophilized formulations in pre-filled syringes, pens, and autoinjectors in dual chamber
disadvantages of lyophilized powders
must be reconstituted prior to injection, less convenient than solutions
more expensive and time-consuming to manufacture
lyophilization
also known as freeze-drying
removes water by sublimation
occurs at low temperature and low pressure
gentler than other methods of removing water (like boiling) so better for fragile drugs
same process is used for some foods (like instant coffee)
lyophilization and instability
instability is caused by lyophilization as “freeze-concentration” can promote aggregation
disulfide bond scrambling can be accelerated by freezing and drying
protein structure can be perturbed by lyophilization
aggregated and/or degraded protein can be observed on reconstitution
role of lyoprotectants and cryoprotectants
stop lyophilization from perturbating the protein structure
clinical considerations of lyophilization
use caution when
- reconstituting
- storing and handling the reconstituted solution
- administering (visually inspect for particulates)
