Case 5 Flashcards
biotechnology
any technique that uses living organisms (bacteria, yeast, mammialian) in the production or modification of products used to affect human health and human environment; macromolecules
biotechnoloy products include
proteins, DNA, RNA, MABs
biotechnology is used to
treat diseases, prevent and diagnose
the first biotech product was
insulin, banting and best, Univ of Toronto; 1912
insulin
comes fro the pancreatic islet cels and insulin is isolated from animals (first cows then pigs), now recombinant human insulin is used
problems with insulin from animals
impurities, allerigies, contaminants
rDNA
recombinant DNA; proteins, MABs
PCR
polymerase chain reaction; proteins, gene therapy, antisense NAs, large scale production
antibody production via hybridoma technology
MABs
cohen boyer method
makes rDNA
step 1 of cohen boyer method
DNA is first cut into smaller lengths with ECORI 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 of the cohen boyer method
rDNA production
protein production begins by incorporating DNA of interest into the plasmid
ligase is used to connect ends of DNA fragment with the ends of the plasmid DNA effectively making a slightly larger circular unit
step 3 of the cohen boyer method
host cell selection and protein production
cloning can then be accomplished by inserting the rDNA into a host that replicates easily; bacteria, yeast, mammal cells,
bacterial hosts example
Ecoli
bacterial host advantages
replicate fast
cheap
simple proteins
bacterial host disadvantages
bacterial debris
pyrogens
antigens
cannot make post translational modifications (glycosylation)
Yeast host cell example
Saccharomyces cerevisiae
advantages of yeast cell hosts
protein secretion fast growth rate large scale production absense of pyrogens not pathogenic post translational modifications
disadvantages of yeast cell hosts
active proteases can degrade proteins
mammalian cell hosts example
chinese hamster ovary cells
mammalian cell host advantages
folding and post-translational modifications
contamination
more complex proteins
mammalian cell host disadvantages
cost
time
polymerase chain reaction
makes genes, proteins, antisense NAs; quick scale up
process of PCR
denaturation (heat up to separate)
annealing (introduce primers) cool
extension (introduce DNA polymerase) hot
post translational modifications
often necessary to obtain a functional protein; glycosylation, proteolytic cleavage of a pro-peptide; disulfide bond formation, protein folding
post translational modifications fall into two broad categories
needed to produce a functional protein and that produce enhanced pharmacokinetic properties
glycosylation
most common post translational modification of proteins; attachement of polysaccharide chains to specific amino acid residues, carbohydrate components may play a variety of critical roles (bacteria hosts cannot do this)
pegylation
attachment to PEG, changes immune system response to them; proteins get hidden from the immune system like native proteins
forms a shell around protein
hinders metabolism
increases circulation time
pegylation example
neupogen to neulasta
half life increases
longer acting
once per chemo cycle admin
immunogenicity of biologics
anti antibody responses (AARs) antidrug antibodies (ADAs)
blockbuster product
$1 billion in sales
1st of its kind
lead for new drug/biologics category
monoclonal antibodies
AAs; controls immune response to foreign materials; administered IV, SQ, IM, low BA and high chance for adverse drug reactions
antibodies
secreted by t lymphocytes
body makes polyclonal but monoclonal means
uniform structure, specific, derived, single clone of cells, specific gene sequence
MAbs
specifically target cell receptors RA, Metastatic breast cancer very specific (leads to small market)
best selling biologic
Humira (adalimumab) RA, CHO cells
MAbs use
CHO but 4 use Ecoli
1st MAb
OKT3 CD3 on Tcells or Orthoclone
latest MAb
Tecentriq
CHO cells
bladder and UT cancer
combination therapy with Abraane for metastatic triple negative breast cancer
Mouse Chimeric Humanized Human which has higher immunogenicity and types associated with each
omab ximab zumab umab human = lowest immunogenicity
toxicities associated with MAbs
MAb antigen binding
mAb interactions with the target antigen on tissues other than the intended target
Fusion Proteins
Enbrel (Etanercept) blockbuster biological product Fusion of EC domain of TNF-a receptor and Fc portion of human IgG1 anti inflammatory disorders CHO cells
classes of biotech products
monoclonal antibodies synthetic immunommodulators recombinant vaccines recombinant hormones recombinant enzymes gene therapy and nucleic acid based engineered cell based purified proteins
growth factors or colon stimulating factors
ESAs (erythropoietin stimulating agents) CSFs (colony stimulating agents) safety concerns small molecule competition regulate process by which stem cells in bone marrow reproduce / differentiate admin: SQ or IV
colony stimulating factors (CSF) Neupogen
not glycosylated
half life 3.5 hous
daily dosing by injection to maintain its effects on bone marrow
Ecoli
Neulasta
pegylation increases size of filgrastim
too large for renal clearance
retains the same biological activity binds to the same GCSF receptor stimulating the proliferation, differentation and activation of neutrophils
15-80 hour half life
longer acting
single dose compares to 11 doses of neupogen
Erythropoietin Stimulating Agents
Procrit/Epogen/Epoetin alfa
half life of 4-13 hours
mammalian cells
Aranesp/Darbepoetin alfa
extra glycoslyation
carbohydrate linked components increased half life
requires glycosylation for biologic activity
CHO = host
cytokines
generally defined as soluble mediators or glycoproteins
communication between cells in the immune, hematological or neurological systems
2 groups: Interferons and Interleukins
Avonex IFN-B; relapsing multiple sclerosis, CHO cells
Vaccines
prophylactic and therapeutic Recombivax HB: Hep B 3 dose schedule has a surface antigen that will stimulate immune response antibody production in the body S. cerevisiae
hormones
insulin and its analogues
administered SQ
Lantus (insulin glargine) market leader in biotech hormones
E Coli
Unique conditions for biotech products
- biosimilars and interchangables
- manufacturing
- storage
- admin
biosimilar
a biotherapeutic product which is similar in terms of quality safety and efficacy to an already licensed biotherapeutic product
highly similar to a US licensed reference biological product nowithstanding minor differences in clinically inactive components and for which there are no clinically meainingful differeces between the biological product and the reference product in terms of safety, purity and potency of the product
a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product
biosimilars
developed using unique biological systems and living cells
analytical non clinical and clinical data needs to be similar to original in terms of structural characteristics, safety and efficacy
requires high investment compared to generics
manufacturing a biotech
techniques used:
rDNA technology
PCR
back in the day - hybridoma technology for MAbs
manufacturing process
large scale manufacturing plants
intensive development work in cell line, media and bioreactor condition optimization, cell specific productivity hae allowed high titers up to 10 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
storage
proteins are significantly more fragile than small molecule drugs
extremes in temp can cause proteins to aggregate or damage 3D conformation
usually stored at 4 degrees
too much heat = denature
dont want to freeze
stabilizing biotechnology products during storage
lyophilization “freeze dried”
a technique that converts an aqueous solution of a protein into a solid
elimination of water use vacuum
use protectants to replace lost water and keep 3D/tertiary structure intact
reconstitution
before use if product was lyophilized most population - use bacteriostatic water for injection USP preservative SWFI for newborns do not use if solution is cloudy WFI = only use each vial once
admin variations for biotech products
therapy - self admin
RA
MS
Psorisasis
Conditions
vaccines
asthma
immune disorders
Chemotherapy
cancer
anemia
neutropenia
self admin
demonstrate techniques to patients let them perform first injection under supervision importance of rotating injection sites - avoid site rxns safe disposal of needles and syringes storage instructions / light do not shake / agitate too much protein roll in the palms of hands importance of regiment compliance
