Quiz 5 Flashcards
biotechnology
technique which uses living organisms (microorganisms) bacteria, yeast, mammalian in the production of products used to affect human health and human environment
uses of biotech
treatment, prevention, diagnostics
size of biotech
macromolecules
biotech products
proteins, Nucleic acids, monoclonal antibodies, RNA
small molecule medicines vs large molecule medicines
small: single chem synthesized, active ingredient, made entirely from chem-synthesized reactions between different compounds, manufactured in a chemical process
large: made from living cells and complex, active ingredients are protiens, antibodies, cytokines, insulin, biologics derived from living organisms, characteristics and properties influenced by the manufacturing process, sensitive to changes in their enviroment and handling (all different final products)
CDER vs CBER
both FDA regulated CDER is for small molecule medicines and CBER is for large molecule medicines
first biotech product
insluin, 1921, university of toronto, banting/best
insulin then vs now
then: isolated from cows and pigs
now: recombinant human insulin
issues with then: animals not all the same, allergies/immune response, containmenents
rDNA
used to produce biologics, proteins, mAbs, developed in 1973, used to obtain large amounts of protein higher level of purity and lower cost
PCR
polymerase chain reaction, proteins, gene therapy, antisense NAs, large scale production if possible
hybridoma technology
antibody production
when was the first rDNA marketed?
insulin - 1982 - FDA approved
how to obtain the biotech product / protien
- isolation of DNA with gene of interest
- insertion into plasmid for protein synthesis - rDNA - independent of nucleus
- host selection for scale-up
cohen-boyer method
1971-EcoRI sed to cut plasmid restriction endonuclease
1972 -insertion of rDNA so foreign DNA will replicate naturally
step 1 cohen boyer method
DNA isolation - DNA first cut into smaller lengths with restriction endonucleases which recognize specific sequences of base pairs and cut the DNA at that point. the DNA sequence desired can therefore be removed and isolated.
step 2 cohen boyer method
recombinant DNA production - protein production begins when incorporating the DNA of interest into the plasmid DNA, DNA segment mixed with the plasmid DNA and ligase, ligase connects the DNA with plasmid, forms the rDNA
step 3 cohen boyer method
host cell selection and protein production - cloning accomplished by inserting the recombinant DNA into a host that replicates easily - bacteria, yeast, mammalian cells, proteins increase in complexity with increasing host complexity
bacterial hosts
advantages: replication rate is rapid, cheap used for simple proteins
disadvantages: bacterial debris, pyrogens, antigens, fever causing not in here!
cannot make relevant post-translational modifications
can do glycosylation
recombinant insulin came from
E. Coli
humalin
rDNA insulin
yeast host cells
advantages: protien secretion, growth rate, large-scale production, absence, not pathogenic, post-translational modifications
disadvantages: active proteases can degrade proteins
yeasts are attractive hosts for the production of therapuetic proteins, used to express recombinant proteins to overcome the shortcomings of bacterial expression systems
example of yeast host
saccaromyces cerevisiae, leukine is a drug
mammalian host cells
some proteins only produced with higher organisms, proteins are difficult to express and need folding complex for function
advantages: folding, post-translational modifications, contamination, more complex proteins
disadvantages: higher cost, more time
example of mammalian host cells
chinese hamster ovary cells, aransep is an EPO produced in CHO
immunogenicity of biolgoics
anti-body responses (AARS)
anti-drug antibodies (ADAs)
pegylation
used to extand half life
filgrastim (neupogen)
half life of 3.5 hours, 18,800 Da, requires daily dosing by injection to maintain its effects in the bone marrow
pegfilgrastim (neulasta)
half life of 15-18 hours, longer acting form, 20KDa PEG molecules to the N-terminal of filgrastim, once per chemotherapy cycle admin
peglation
earliest chem modifications of therapeutic proteins, attachment to PEG, conjugation of proteins to PEG changes the immune response to them
result- proteins get hidden from the immune system like native proteins, forms a shell around protein, hinders circulation time and metabolism
polymerase chaine reaction
makes genes, protein, antisense NAs, quick scale up
- denaturation
- annealing
- extension (2 minutes 72 degrees C- only dNTPs
denaturation
DNA heated which allows the strands to separate/denature(1 min 94 degrees C)
annealing
single-stranded DNA (primers) are added. these bind to complementary sequences of DNA interest, bracket the region to be replicated (45 seconds 54 degrees C)
extension
DNA Polymerase is added, DNA heated again. DNA polymerase added, DNA heated again. DNA polymerase starts at primer and synthesizes few DNA complementary to the single strand - developed 1983 - very quick method for replication
post-translational modification
often necessary to obtain a functional protein. occurs through- glycosylation, proteolytic cleavage of a pro-peptide bond, disulfide bond formation, protein folding
post-translational modification falls into 2 broad categories
- those needed to produce a functional protein- glycosylation
- produces enhanced pharmacokinetic properties - glycosylation/pegylation
glycosylation
most common post-translational modification of proteins - attachement of a polysaccharide chain to a specific AA residue. carbohydrate components may play a variety of critical roles
how do you glycosylate a recombinant protein?
easiest system for recombinant protein - E Coli BUT resulting protein not glycosylated (E. Coli lacks an endogenous glycosylation pathway)
conditions unique to biotech products
- biosimilars and interchangeables
- manufacturing
- storage
- administration
definition of a biosimilar
basically a generic form of a biotech product
WHO: similar quality, safety and efficacy to an already licensed biotherapeutic product
USFDA: no meaningful differences, same in safety, purity, and potency
EMA: version of active substance of an authorized original biological medicinal product
Can large molecule medicines be exactly reproduced?
no - derived from living cells. can be highly similar to the orginial in terms of safety, purity, and potency and used to treat the same illness. AA sequence is expected to be the same as the reerence product
what data needs to be similar to the original biologic
analytical, non-clinical, clinical data (characteristics, safety, efficacy) - more expensive and more involvement with patients - require high investment (clinical trials and pot-approval safety monitoring)
zarxio
first biosimilar approved in the US and added to the FDAs new purple book - march 6th 2015 - biosimilar product of amgens cancer drug neupogen - approved 2015 after launch of purple book 2014
herceptin
trastuzumab, fourth tratuzumab biosimilar granted FDA approval
purple book
lists of licensed biological products with reference product exclusivity and biosimilarity or interchangeability evaluations - adding in a list to describe the degree to which a biosimilar drug is equivalent to the biologic product. launched in anticipation of biosimilar product zarxio
manufacturing a biotech
rDNA technology
PCR
back in the day - hybridoma technology for MAbs (mouse + protein to secrete antibodies and extract the spleen to get B -lymph - so lymph + mylenoma cells
manufacturing process
large scale plants - multiple 10,000 L or larger cell cultures bioreactors (fermentation tanks)
intensive development work in cell line, media, bioreactor conditon g/L and cell densities of over 20 million cells/mL in fed-batch processes to be achieved - all biotech products NEED to be sterile with optimal growth condtions (temp, O2/ nutrients/pH)
storage
proteins more fragile than small-molecule drugs
extremes in temp can cause proteins to aggregate or damage 3D conformation - stored at 4 degrees - too much heat will cause denaturation (unfold/break down)
lyophilization
“freeze dried” converts aqueous solution of a protein into a solid - elimination of water, use a vacuum conditions, protectants to replace lost waste and keep 3D/ tertiary structures in tact
reconstitution
before use if product was lyophilized - use bacteriostatic water for injection - has a preservative - in newborns use WFI (use each vial only once) - do not use solution of cloudy
admin for biotech product - therapy
self - admin
RA, MS, psoriasis
admin for biotech products - conditions
injected in a clinical setting - vaccines, asthma, immune disorders
admin for biotech products - chemotherapy
under trained medical personnel - cancer, anemia, neutropenia
self admin
demonstrate reconstitution, perform first injection under supervision, rotating sites to avoid site reactions, safe disposal of needles and syringes, storage instructions/light, do not agitate, roll in palms, regimen compliance
top biotech products marketed (biggest to smallest)
monoclonal antibodies, synthetic immunomodulators, vaccines, recombinant hormones (insulin, NPH, lantus), purified proteins, recombinant enzymes
$192 billion total value including drugs in development
blockbuster product
drugs that make a lot of money, $1 billion in sales, 1st of its kind, lead for new drug/biologics category, Humira (IV, SubQ)
orphan drug
treats a small population of people
ex: cystic fibrosis - manufaturing companies do not want to invest in these disease states
how many MAb on market
110
Hinge
helps with flexibility
VH
variable heavy chain
VL
variable light chain
Fab-Fragment
binds the antigen
FC fragment
crystallizable fragment - helps stay in circulation longer and stabilizes the fragment
monoclonal antibodies
made of of AA, bone marrow helps to produce antibody (immunoglobin secreted by B cells)
controls immune response to foreign materials, antigen stimulates the antibody production, IGG change variability of the binding sites FV
administration of monoclonal antibodies
IV, SQ, IM (SQ and IM are fragile)
issues- BA and adverse drug infusion reactions (BC protein )
antibodies
secreted by the B lymphocytes, body makes polyclonal,
monoclonal
uniform structure, specific, derived, single clone of cells, specific gene sequence
treatment of MAbs
targets receptors on cells, very specific, specificity is a disadvantage because market for these drugs can be small, adv: less adverse reactions because so specific
Humira
adalimumab, RA (anti-inflammatory, MAb, 2002 approved, binds to TNFalpha - anti-TNF alpha, higherst revenue biologic on the market, Abbvie, CHO
herecebtin
trastumab, 1998, HER-2 overexpressed receptor
first Mab approved
1986, muromomab, recruits t-cells, binds to CD3 on the t-cells, alled OKT3 or orthoclone, prevented kidney transplant injection
how many FDA biologics/biosimilars approve as of 2018
100 FDA approved
amjevita (adalimumab)
2016 FDA first approved generic for humera, amgen, CHO, hard to get same product because it is a protein and it has a 3D structure, may not be the same but it does the same thing
MAbs host cells used
E-coli, CHO, Mammalian
biotechs in mammalian
MAbs, biospecific (2places for attachment of antigen), antibody drug conjugates, radio-labelled (isotope lit up), antigen binding fragment (Fab), fusion proteins)
biotechs in Ecoli
fusion proteins, Fabs
latest Mab
tecentriq (atezolizumab) FDA approved 2019, genentech, CHO cells bladder and UT cancer, combination therapy with abraxane for metastatic triple negative breast cancerwhich expresses PD-L1
mouse MAb
mo = mouse
ex: muromomab, high potential for immunogenicity
chimeric MAb
Xi = 75% human, rest mouse (better acceptance than mouse)
ex: rituximab
humanized MAb
zu = human, more acceptance than chimeric
ex: trastuzumab
human MAb
mu, su= fully human
ex: adalimumab, low potential for immunogenicity
toxicities with MAb - target related
- mAb binding specific - HUMIRA - may cause immunosuppression and lead to infection
- mAb interactions with the target antigen on tissue other than the intended target- anti EGFR mAb causes skin rash, pruritus, erythema - EGFR target receptor that reflects the wide expression of epideraml growth but also on skin cells
toxicities with MAb - modality related
target-independent and can occur acutely at the time of injection, or develop through prolonged treatment with the antibody,
acute immune reactions- hypersensitivity reactions, cytokine-release syndrome, infusion-related reactions
fever, nausea, chills, cytokine release,
enbrel (etanercept)
fusion protein, blockbuster biological product - fusion of extracellular (EC) domain of the TNF - alpha receptor and Fc portion of the IgG1, anti-inflammatory disorders, CHO cells
fusion proteins admin
pre-filled syringes - SC
synthetic immunomodulators
cytokines - 20
growth factors - 35
growth factors/ colony-stimulating factors
ESAs (EPO stimulating agents) (erythrocytes/ leukocytes) and CSF (colony stimulating factors (WBC)), regulates process by which stem cells in bone marrow reproduce/differentiate
overall decline in sales of ESA
safety concerns and small molecule competition
admin of growth factors/colony-stimulating factors
IV or SC
neutropenia
neupogen (fibrostin)
neulasta
anemia
epoetin alpha, poeitin alpha
neupogen
colony stimulating factor
filgrastin, not glycosylated, half life 3.5 hours, E-coli host, requires daily dosing by injection to maintain effects on the bone marrow
neulasta
CSF, pegylation increases size of filgrastim, too large for renal clearance, retains same biological activity, binds to same G-CSF receptor, stimulates proliferation, differentation, activation of neutrophils - same AA as neupogen just addition of PEG, half life 15-18 hours - single dose not cheaper
procrit/epogen/epoetin alfa
parent biologic, stimulates RBC, half-life 4 to 13 hours, glycosylated protein, mature polypeptide 165 amino acids, mammalian cells - requires glycosylation for biologic activity - CHO host
aranesp/ darbepoetin alpha
difference is carbs, parent + glycosylation, same AA just diff carbohydrates, half life 27-89 hours, 5 N-linked oligosaccharide chains, no change in tertiary structure/biologic activity
requires glycosylation for biologic activity - CHO host
cytokines
lymphatic system
soluble mediators or glycoproteins, helps communicate between cells in the immune system, hematological or neurological systems, interferons/ interleukis, avonex IFN-beta, relapsing MS, CHO host cells
prophylatic vaccine
proteins for future pathogens
therapeutic vaccines
after the infection has occured
recombivax HB
hep B (energix-B, recombivax HB), 3 dose schedule, has surface antigen that will stimulate the immune response (antibody production in the body) - S. cerevisiae
provenge
therapeutic prostate cancer vaccine - personalized therapy- stimulates patients’ immune cells outside the body with PAP and GM-CSF - 93,000
