Bio tech (EXAM 3) Question set 3- Biopharmaceuticals (Wyatt pg 27-60), – Flashcards
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purpose/function of 2 extra agents in denaturing gel
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1) beta-mercaptoethanol: used to break disulfide bonds linking cysteines together 2) SDS:detergent that disrupts all other non-covalent bonds (H bonds, van der waals, etc)
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what are biopharmaceuticals?
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*polypeptides* that are used as medicines- forming huge molecules for therapeutic use
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types of biopharmaceuticals
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-hormones: hematopoietic, wound healing, growth -cytokines: act in inflammation/immune sys -enzymes/ERT -antibodies
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why use biopharmaceuticals?
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able to have a treatment option that is otherwise not possible with small molecules or gene/stem therapy yet
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gene therapy in relation to biopharmaceuticals?
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-gene therapy: ultimate delivery of protein therapy--> bc genes tell cell to make the specific protein of interest
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biopharmaceuticals are *selective* in hormone/cytokine therapy because?
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protein interactions are more *specific* and with higher *affinity* than a small molecule with their targets, which makes protein therapy harder
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ERT (enzyme replacement therapy) in order to?
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-catalyze rxn otherwise not possible -as a result of genetic/biochemical defect causing a pathological problem
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limitations to biopharmaceuticals
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-require injection (can't give protein orally bc will be degraded by GI system) -instable, degrade and clearance fast -expensive
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biosimilar
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-acknowledged by ACA in an attempt to get "generics" for biopharmaceuticals -must be only "highly similar" to already approved biological product, but not entirely simiar
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number of in-process tests done for biopharmaceuticals vs chemical drugs?
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bio: 250 in-process tests chemical: 50 in-process tests = more time verifying biopharmaceuticals
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problem with biosimilar only being "highly similar"
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-may cause ADR that brand didn't -leading to immune reaction to original brand drug if switched back? -difficult in ensuring proteins are the same because they are so large and complex
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EPO glycosylation example?
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-turned EPO (endogenous protein) into a biopharmaceutical -normally has 3 N-linked glycosylations and 1 O-linked, but where these were different in the biopharmaceutical it caused ADR in pts receiving it
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biobetter
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protein with a *non-natural* modification(s) that improves the pharmaceutical properties of the original protein =adding something to change the protein and its ADME
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biobetter ex- PEG
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adding PEG groups on proteins as drugs to protect from degradation/enhance solubility, etc
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Cimzia
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already approved biopharmaceutical with non-natural modifications (PEG) to improve drug
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steps to develop biopharmaceutical
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1) isolate RNA from human cells that already expresses the mRNA 2) make double stranded cDNA 3) ligate to vector (plasmid) 4) transfer into host cells so host cells make the protein 5) purify protein from host cells to get protein of interest
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types of host cells that can express foreign proteins?
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-bacterial (E.coli) -yeast -mammalian -carrot root cells
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what happens to vector/plasmid in mammalian host cells?
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gets incorporated into host genome
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advantages of bacterial/yeast host cells
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-easy to grow -higher yield -cheaper than mammalian -plasmids easy to introduce into bacteria or yeast
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disadvantages of bacterial/yeast host cells
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-limited post-translational modifications (bc yeast doesn't do same modifications as humans do) -protein misfolding and solubility (don't have ER chaperones)
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advantages of mammalian host cells
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-do post-translational modifications (*glycosylation*) -proper folding (w/ ER chaperones)
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disadvantages of mammalian host cells
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-difficult to isolate cells that overexpress protein of interest -higher cost -lower yields
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why is purity a concern?
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bc don't want any residual bacterial protein in injection- could cause immune reaction in pt
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after protein is purified what happens?
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must be: -properly folded -catalytically active, if in ERT
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3 ways proteins differ according to FDA
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1) primary AA sequence 2) modification to AA- glycosylation, etc of side chains 3) high order structure- folding
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hetergenetiy in biopharmaceuticals?
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can happen when there are *modifications to AA* = difficult to control and variable activity
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protein modifications/higher order structure can be affected by?
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*environment* -light -temp -moisture -packaging (due to RPh handling them)
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3 techniques to separate and purify proteins
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1) chromatography 2) gel electrophoresis 3) dialysis
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gel filtration chromatography purpose
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separates proteins by *size* (aka size-exclusion chromatography)
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how does gel filtration chromatography work?
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1) polymer beads/gray spheres covalently linked to column 2) load protein mix 3) flow solvent/buffer through -small proteins: enter polymer beads via pores & are held back -large proteins: pass around beads and are able to collect first
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fraction collecter
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at the bottom of the gel filtration chromatography to collect small volumes of liquid flowing through
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ion exchange chromatography purpose
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separates protein by *charge*
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how does ion exchange chromatography work?
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1) polymer beads have a net negative or positive charge 2) AA are negatively or positively charged and the ratio affects overall net charge and pass through channel via repulsion forces -negatively charged beads: negatively charged proteins pass -positively charged beads: positively charged proteins pass
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how to get remaining proteins off beads in ion exchange chromatography?
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bound with *non-covalent* interactions and can increase NaCl concentration to push remaining proteins off
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ex of negative charged bead
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carboxymethyl group (ionized form)
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ex of positive charged bead
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diethylaminoethyl group (protonated form)
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affinity chromatography purpose
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separates proteins *bound by specific molecule* -impt in monoclonal antibodies
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how does affinity chromatography work?
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1) bait molecule is attached to polymer beads- covalently linked 2) protein of interest binds to bait molecule (ex- Glucose) and all other (waste) proteins filter through 3) add more bait molecules to chase off protein of interest (the one that binds to glucose-ex)
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purpose of gel electrophoresis
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-to separate proteins w/ acrylamide gel + buffer solution -matrix acts as sieve to separate proteins (with +/- charges pulsing)
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technique of gel electrophoresis
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1) prepare protein samples (add color dye) 2) load protein samples in gel 3) connect wire and turn on power to make negative and positive poles 4) proteins of different sizes move across matrix at different speeds -smallest: travel farther bc can move easily 5) stain gel to visualize proteins (*last*)
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representative gel in gel electrophoresis?
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-compare protein of interest to "standard/ladder" of proteins with known size
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how do proteins migrate in gel electrophoresis?
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top to bottom -able to see lines/different sizes and densities
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acrylamide
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-most common gel used in electrophoresis -able to change concentration depending on protein being analyzed
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when to use higher % acrylamdie?
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the *smaller the protein* use higher % gel (bc the higher % has a tighter sieve)
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native (non-denaturing) gel?
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used to prevent unfolding of protein and to separate proteins based on *size*
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denaturing gel?
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contains 2 extra agents in the buffer to denature protein 1) beta-mercaptoethanol 2) SDS -included in preparation before loading sample in gel to break up higher order structure (2, 3, 4)
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non-denaturing vs denaturing gel purpose?
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non-denaturing: separates by *shape* denaturing: separates by *molecular weight/mass/number of AA*
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dimers on non-denaturing vs denaturing gel?
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dimer would look bigger on non-denaturing bc it is separated by *shape*
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Lane 1 in protein purification
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contains homogenate/crude cell isolates with many different proteins
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Lane 2 in protein purification
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salt fractionation- simple step in purification to precipitate out unwanted proteins
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Lane 3-5 in protein purification
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show the results after 3 chromotagraphy purification steps
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what happens in each step of chromatography?
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1) unwanted contaminate proteins are decreasing 2) protein of interest is increasing in *density* (intensity of band shows how much protein is in that sample)
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types/ex of hormones used for biopharmaceuticals
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-hematopoietic (EPO) --insulin
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dialysis (protein purification)
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1) using dialysis bag- put concentrated solution from host cell 2) small particles (glucose/bait) will diffuse out of bag- while larger proteins (of interest) won't 3) repeat steps to filter out smaller/unwanted particles until left with only protein of interest in bag
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evaluation of success in protein purification (3 ways)
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1) specific activity (measure activity of enzyme in mg or mcg) 2) yield- how much purified substance you get 3) integrity- if protein is properly folded, etc
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what happens to protein yield and specific activity after multiple purification steps?
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-protein yield *decreases*- bc losing more proteins and only gaining protein of interest -specific activity *increases* bc getting more of active protein
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Example 1 of biopharmaceutical case study (hormone)
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insulin
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type I diabetes caused by?
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-autoimmune destruction of beta cells that produce insulin --> use isolated insulin (from pigs --> humans) to treat
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structure of human pro-insulin
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A + B chain linked with disulfide bonds (3) -C (aka connecting) peptide (must be cleaved)
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why initially used pork insulin?
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bc sequence of AA was only one AA different than human insulin
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steps of initial production of human proinsulin
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1) isolate mRNA from beta cells in pigs 2) produce double strand cDNA 3) cDNA ligated into plasmid 4) put into bacteria to produce proinsulin 5) isolate from bacteria to convert to insulin
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why can't produce insulin in bacteria?
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bc human insulin must undergo post-translational modifications =need to give bacteria cDNA of exact copy of human insulin -biotech companies then do post-translational modification and cleave C-peptide to give active insulin
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essential post-translational modifications in insulin?
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-cut out introns -cleave C peptide with protease
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2nd generation human insulins?
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bioengineered- biobetter
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what was different with 2nd generation insulin
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-contained non-native amino acid changes to accelerate (rapid acting) or prolong (long actin) duration of action
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Example 2 of biopharmaceutical case study (hormone)
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EPO
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EPO (Erythropoietin)
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natural hormone produced in kidneys to stimulate RBC growth
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kidney failure causes?
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anemia= lack of EPO
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Epoetin Alfa (Epogen, Procrit)
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-EPO made into BioRx -first to treat anemia w/ chronic renal failure, now to tx anemia from many conditions
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how Epoetin produced?
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1) EPO cDNA discovered and cloned into mammalian vector 2) transfected into host cells (CHO) 3) cDNA was integrated into genome of host cells 4) host cells transcribe EPO --> protein 5) EPO *glycosylated* and secreted by host cells = harvested, purified, and formulated into drug
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essential post-translational step in EPO production?
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glycosylation
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difference in post-translational modifications in insulin vs EPO
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-insulin: biotech company does them (cleaves C peptide) -EPO: host cell does them (mammalian- CHO)
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glycosylation of normal EPO
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-one O-linked (Serine) -three N-linked (Asparagine)
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Darbepoetin (Aranesp)
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*bioengineered Epoetin* -two extra glycosylation sites to improve 1/2 life in blood -tx: cancer related anemias & *kidney pts not on dialysis*, but recommendations for lower doses
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why want to improve 1/2 life of biopharmaceuticals (esp EPO)?
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bc they are proteins- and rapidly degraded in body -and to decrease amount of injections
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Example 3 of biopharmaceutical case study (cytokine)
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Cytokine ex- Colony Stimulating Factors
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CSF (colony stimulating factors)
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stimulate cell growth -ex: granulocyte- CSF
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Pegfilgrastim (Neulasta)
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*Biopharm version of granulocyte- CSF* -to treat neutropenia (low white blood cell count) -in E.coli cells -added PEG group (after purifcation) to increase half-life and decrease dosing frequency
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Example 4 of biopharmaceutical case study (Enzyme)
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t-PA
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how does FDA measure pharmacological activity of protein (BioRx)?
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*functional assays* -evaluated function of protein in vitro and/or in vivo -must be able to define specific activity
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t-PA (tissue plasminogen activator)
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protease, when *in presence of fibrin* (forms blood clots), converts the enzyme plasminogen to *plasmin*- to break down fibrin in blood clot
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Alteplase (Activase)
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*biopharm version of tPA* -CHO produced -full length t-PA -tx: early signs of MI/stroke
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process of producing Alteplase
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1) isolate cDNA for natural human tPA 2) put into vector (plasmid) 3) integrate into host genome (CHO cell) 4) form protein and extract/purify protein
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Reteplase (Retevase)
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*biopharm version of tPA* -E.coli produced -*truncated version* & non-glycosylated = increases half-life -tx: early signs of MI/stroke
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process of forming Retplase
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1) isolate from E.coli 2) refolded by biotech company
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Tenecteplase (TNKase)
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*biopharm version of tPA* -CHO produced -modifications to natural cDNA (full length) to produce that make it more *specific* in presence of fibrin= decreases side effects -tx: early signs of MI/stroke
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Tenecteplase modifications to human tPA
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-allows it to be more specific *in presence of fibrin* (less active w/o fibrin) =better management/less side effects
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Example 5 of biopharmaceutical case study (enzyme replacement therapy-ERT)
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Pompe disease- alpha-1,4-glucosidase
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Pompe disease
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-due to lack of alpha-1,4-glucosidase (catalyzes breakdown of glycogen) -leads to lysosomes swelling with glycogen =cardiorespiratory failure before 2 yrs old
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Alglucosidase alpha (Myozyme)
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*recombinant human alpha-1,4-glucosidase* -CHO produced -tx: Pompe disease -biotech companies must expose terminal mannose after extraction from CHO cells
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why Myozyme needs to have terminal mannose (sugar)?
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to be able for body to take up drug in lysosomes and accumulate in lysosomes for action to occur
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what haplotype of alpha-1,4-glucosidase is produced as Myozyme?
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*most predominate*
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Lumizyme
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Myozyme made on larger scale (had to change name, and changed glycosylation patterns)
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ADR of pompe disease drugs (Myozyme & Lumizyme)
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-black box warning for possible 1) immune reactions --> must be in hospital to monitor with first dose & infuse over 4 hours 2) cardiorespiratory failure -increased risk of infections
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Pharmacy considerations of children w/ Pompe disease
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-don't want too sedated -vent management/pulmonary meds -rule out infectious causes -intubated for nutrition -evaluate meds due to impaired renal function
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preparation of Myozome
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-no shaking (protein) -avoid foaming -mix in appropriate volume (take air out bag)
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admin of Myozyme
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-infuse over 4 hours -monitor vital signs
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monitoring w/ Myozyme
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-check: IgG antibodies (every 3 months for 2 years, then annually) -liver function tests -vital signs -volume overload
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pharmacy care (pulmonary)
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-RSV prophylaxis (vaccine) -Albuterol
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Pharmacy care (cardio)
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Enalapril
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Pharmacy care (FEN/GI)
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-ergocalciferol -PEG (laxative) -formula for feeds
