Lectures 19/20

Question 1

biologic drugs

Answer 1

medicinal agents derived from living systems, in contrast to more conventional “small molecules” drugs produced by chemical syntehsis

Question 2

types of biologic drugs

Answer 2

recombinant proteins
plasma immunoglobulins (IVIG)
some peptides
vaccines
DNA
RNA, mRNA
cell and gene therapies
cell-based therapies
AAV vectors
gene editing (CRISPR)

Question 3

peptides and oligonucleotides (tides)

Answer 3

have properties intermediate between small and large molecules
also included in biologics

Question 4

common features of biologics

Answer 4

biological origin. (animal or cellular/molecular biology)
high molecular weight
higher order structure

Question 5

early biologics – 1920s

Answer 5

making insulin in the 1920s through extraction of porcine insulin from pig pancreas at Eli Lilly
high sequence homology among human, porcine, bovine insulin

Question 6

early biologics – 1957

Answer 6

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

Question 7

recombinant proteins in the 1980s

Answer 7

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

Question 8

recombinant proteins (mAbs)

Answer 8

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

Question 9

CAR-T

Answer 9

approved in august 2017
chimeric antigen receptor (CAR) T-cel therapy
examples – Kymriah, CTL019, Tisagenlecleucel, Novartisc
contrast – allogeneic cell therapy

Question 10

Kymriah

Answer 10

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

Question 11

mRNA vaccines

Answer 11

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

Question 12

LNP

Answer 12

contains four types of lipids that condense mRNA and enable delivery to the cytoplasm

Question 13

characters of biologics

Answer 13

DNA
mRNA
cells
proteins
viral vector
animal source

Question 14

biologics timeline

Answer 14

early biology – isolated from animal tissues
recombinant proteins – expressed in transfected cell lines; act as protein factories

Question 15

role of cells in biologics

Answer 15

protein factory
therapeutic agent (ex. CAR-T)
therapeutic target

Question 16

role of viral vectors in biologics

Answer 16

transfection reagent
therapeutic agent
therapeutic target (ex. COVID-19)

Question 17

role of animal tissue in biologics

Answer 17

flu vaccines (chicken)

Question 18

role of proteins in biologics

Answer 18

therapeutic agent
therapeutic target (receptor)

Question 19

role of DNA in biologics

Answer 19

instructions for transfected cells
therapeutic agent (ex. DNA vaccines)

Question 20

why is biologic dosing usually parenteral?

Answer 20

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

Question 21

why are solution formulations popular?

Answer 21

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

Question 22

clinic concerns for biologics

Answer 22

efficacy
sterility
side effects (dose limiting immune response)
pain on injection (volume administered, pH, tonicity ionic strength)

Question 23

formulation concerns

Answer 23

all clinical concerns and
stability
solution viscosity and ease of injection (can be affected by API concentration)
manufacturability (cost, manufacturing time)

Question 24

factors of stability

Answer 24

aggregation
chemical stability (deamidation, oxidation)
shelf-life
storage conditions

Question 25

formulation variables

Answer 25

solution properties (pH, ionic strength tonicity, drug concentration, volume and exicipients) container, closure (materials) storage conditions (fridge vs room vs frozen)

Question 26

pH variable

Answer 26

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

Question 27

concentration formulation variables

Answer 27

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

Question 28

higher concentration = ? aggregation

Answer 28

greater aggregation thus greater turbidity

Question 29

protein aggregation mechanisms

Answer 29

chemical reaction colloidal interactions unfolding (highest energy)

Question 30

partial unfolding at air/liquid interface

Answer 30

is worse with agitation especially prominent at three-phase boundary (container, air, solution formulation)

Question 31

stabilized protein structure

Answer 31

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

Question 32

de-stabilized protein structure

Answer 32

cosolvent surrounds protein in preferential binding can lead to denaturation

Question 33

erythropoetin

Answer 33

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

Question 34

pure red cell aplasia (PRCA)

Answer 34

can result in sudden onset anemia, death in europe, increased incidence of PRCA has been associated with a change in container closure (stopper)

Question 35

what if the solution formulation doesn't work?

Answer 35

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

Question 36

practical considerations for solution formulations of biologics

Answer 36

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

Question 37

covid mRNA vaccine

Answer 37

suspensions of lipid nanoparticles (LNP) in solution which the mRNA being inside of it mRNA is not active without LMP

Question 38

what is prefilled syringes, pens, and autoinjectors?

Answer 38

medical devices not formulations the solution inside it is a formulation so together it is called a combination product

Question 39

advantages of pre-filled syringes, pens, and autoinjectors

Answer 39

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

Question 40

disadvantages of pre-filled syringes, pens, and autoinjectors

Answer 40

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

Question 41

pre-filled syringe components

Answer 41

syringe barrel luer lock finger grip plunger gasket top cap

Question 42

location of leur lock

Answer 42

in between the top cap and the syringe barrel

Question 43

components of insulin pens

Answer 43

pen cap outer needle cap inner needle cap needle protective seal rubber seal insulin reservoir dose window dosage knob injection button

Question 44

common features of pre-filled syringes, autoinjectors, and pens

Answer 44

drug solution needle piston/plunger housing

Question 45

special concerns for pre-filled syringes, autoinjectors, and pens

Answer 45

higher surface to volume ratio lower total volume syringe lubricants/oils

Question 46

addition of syringe lubricants or oils

Answer 46

change interaction of proteins with solid surface (hydrophobic lubricant lines container) droplets in solution (provide additional hydrophobic surface area)

Question 47

advantages of lyophilized powders

Answer 47

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

Question 48

disadvantages of lyophilized powders

Answer 48

must be reconstituted prior to injection, less convenient than solutions more expensive and time-consuming to manufacture

Question 49

lyophilization

Answer 49

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)

Question 50

lyophilization and instability

Answer 50

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

Question 51

role of lyoprotectants and cryoprotectants

Answer 51

stop lyophilization from perturbating the protein structure

Question 52

clinical considerations of lyophilization

Answer 52

use caution when - reconstituting - storing and handling the reconstituted solution - administering (visually inspect for particulates)