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26 Cards in this Set
- Front
- Back
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What were the limitations of protein based pharmaceuticals and how have they been made more practical? |
Used to be hard to collect, limited quantity, expensive to produce. Now with recombinant engineering, genetic pathways can be engineered into easier to grow microbes so they can produce desired products more quickly and efficiently. Can also allow for greater ease in harvesting product. |
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What are the five major steps to producing protein based medicines for humans? |
Identifying the protein and specifics about it using proteomics. genetically alter microorganism to create desired product in desired culture and release it. Optimize productions from here Extract and purify the protein evaluate biological activity!!!! optimize stabilization and storage to preserve biological activity |
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Why would you want to have various cell banks? |
So you have multiple back ups if something happens. Also lets you study genetic stability of the transgene and keep varying levels of activity in storage. |
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Process flow chart |
get genetic material starting product --> fermentation (chemostat or batch) --> harvest material --> purification |
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What are some potential contaminants for extracted product from industrial scale protein production? |
dead cells, fused proteins, waste products, substrate, truncated forms |
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How could you get a target protein out of an impure substance? |
program in features to separate it like hydrophobicity, use an affinity binder (antigen, glycoprotein, lectin, etc) to separate it, change the electrical charge. Combine it with another non-deactivating molecule so it precipitates out, filter it out, use physical or chemical changes to remove it from the substance |
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What are some of the potential issues with contaminants? |
Could be toxic, heavy metals, mutagenic or oncogenic, change the pharmacological activity of the target protein, be infectious, etc. |
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What are some issues with protein based drugs? |
could be antigenic (causing allergic reactions) or could have stability issues (low half life, in vitro issues, delivery issues) |
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Inspecting the pharmaceutical systems: what do you look for? |
Documentation of Everything!!! material sources, conditions, material types, safety measures, equipment, etc. |
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What are monoclonal antibodies and why are that the largest portion of the market? |
They are used in a huge variety of diagnostic tests and even in treatments (to deactivate a protein) |
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How could you isolate the protein for identification and cloning? |
Do cDNA genesis, clone into another vector and test for expression.
Could do DNA shuffling to optimize |
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What are some diseases that can be treated with rDNA derived proteins? |
AIDS, cancer, cardiovascular disease, rheumatoid arthritis, cystic fibrosis, hemophilia, |
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Vectors used in gene therapy trials? What is gene therapy? |
Gene therapy is altering of genetic information or addition of new abilities - use retroviruses, adenoviruses, and naked/plasmid DNA |
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What is a retrovirus? |
contains reversetranscriptase to allow it to go from mRNA to DNA and integrate with existing DNA. Down side is cells need to be dividing for proper transduction to take place. |
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What is an adenovirus? |
don't actually integrate into the DNA |
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How could the virus replication be used to target cancer cells? |
recombinant viruses could lack a protein inhibiting the protection response of a normal cell. This protection response might not be present in a cancer cell though, so the oncolytic virus would travel in and lyse the cell. Issues? immune response |
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Prodrug - what is and how could it be used? |
A prodrug is something that is transported to the specific location and the activated there by some sort of environmental activator. Typically a drug attached to a protein. |
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Single Stranded oligonucleotides. What are some way nucleic acid therapeutics could be used? |
They are single strands that can knock down gene expression by binding up the transcription factors, matching up to mRNA's to degrade them, inhibit translation or more, could alter ribozymes to degrade mRNA. Great for expression disorders!! |
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What are antisense oligodeoxynucleotide therapies? |
use small synthetic molecules that are similar to single stranded DNA. work just like single stranded oligonucleotides for gene inhibition, drug specificity, and more. Target protein targets. They can bind right to the DNA and block transcription, including during RNA synthesis. can block interaction of mRNA with ribosomes. Can create triple stranded DNA. hard to get across membranes and stability issues |
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How can you increase the stability of antisense oligodeoxynucleotides? |
Change the type of backbone (methylate or phosphorylate), coadminister with cationic lipids, create chimeric molecules. |
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What is an siRNA? |
small interfering RNA's that work with ribozymes and can target many RNA's and essentially cleave them. |
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How do siRNA's work? |
They are synthesized then transfected into cells. they cleave mRNAs |
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What are ribozymes? |
They bind to mRNA and facilitate hydrolysis (cleave the mRNA) |
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how could you use ribozymes to help with pathogenic mRNA illnesses? |
Create a ribozyme that targets the specific sequence and circulate blood outside the system to expose it to the recombinant mRNA plasmid so transduction can occur |
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What is the potential for gene therapy? |
Gene therapy could allow you to alter the genetic make up of individuals so they essentially cure themselves of the symptoms |
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What are some techniques for gene replacement therapy and how well have they worked? |
use of viruses for transfection of DNA. Viruses must be defective so they don't further replicate, and go to the right place. They're very immunogenic
Gene guns have also been used to inject raw DNA.
liposomes (fatty bodies) have also been used to transduce DNA but low take rate and transient improvement. |
