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Recombinant Pharma 1
Biopharmaceuticals 1
| Question | Answer |
|---|---|
| What is biotechnology? | the use of living things like cells and bacteria in industrial processes. |
| What are biopharmaceuticals? | Medicine and drugs that are produced using biotechnology |
| What is pharmaceutical biotechnology? | biotechnological manufacturing processes of pharmaceutical products |
| What is meant by recombinant DNA technology? | The manufacturing of a product via genetic mutation. A plasmid or viral vector is inserted into a suitable micro-organism or cell line to produce a specific DNA code, which after translation into a protein is extracted and purified. |
| What is a vector? | A vehicle (like a plasmid) used to transfer genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism. |
| What is a plasmid? | Plasmids are fragments of double stranded DNA that typically carry genes and can replicate independently from chromosomal DNA. |
| Why are plasmids important? | Easy to work with, self replicating, stable, functional with many species and have a diverse set of applications. |
| What needs to be considered when manufacturing proteins for therapeutic use? | Issues related to manufacturing, purification (often the most expensive part of the process) and characterisation of cels. Biotech products for parenteral route have strict requirements to meet. |
| What are the two methods that proteins can be produced? | Expression systems (transgenic animals and plants) or cultivation systems. |
| Describe how transgenic animals are used to create proteins for therapeutic use | Foreign genes are introduced into animals The proteins is expressed in the animal milk where it can be extracted and purified. Advantage: cheap method of producing proteins in large quantities Disadvantage: animal health |
| Describe how plants are used to create proteins for therapeutic use | Plants have the potential for the desired protein to be expressed in edible seeds like rice. Issues with this method are stability and phenolic oxidases. |
| Describe how cultivation systems are used to create proteins for therapeutic use | The cells are grown in a bioreactor tank at specific and controlled pH, temperature and oxygen tension in a medium of nutrients. Nutrient examples include sugars, fats, sterilised water, amino acids, electrolytes, vitamins, serum, trace minerals, hormones |
| How is the final medium for cultivation systems prepared? | Compononents are dissolved in purified water before sterile filtration. The protein then needs to be filtered/seperated from contaminents in the tank (viruses, endotoxins, metals, purification reagents etc) |
| What is downstream processing? | The recovery of a biolofical reagent from a cell culture supernatant. |
| List the 3 separation techniques used for downstream processing | Filtration/Centrifugation (separation based on density) Precipitation (separation based on solubility) Chromatography (separation based on charge, size, bond type etc) |
| Describe filtration/centrifugation in downstream processing | Products are separated from the biological systems that contain suspended particulate material including whole cells, lysed cells, and fragments of broken cells. How much it costs and how effective it is depends on the particulate material and product |
| Describe precipitation in downstream processing | The method of separation is based on a change in solubility. The solubility of a particular protein depends on the physico-chemical environment (pH, ionic species and ionic strength of solution). |
| Describe how chromatography is used in downstream processing | Separation mainly based on differences in distribution between two phases (solid vs stationary). Can manipulate charges, size of protein, lygand substrates, hydrophobicity and covalent/noncovalent binding to separate. |
| What needs to be considered when formulating biotech products? | 1. Excipients used (especially in parenteral products) 2. Microbes 3. Shelf life of protein based products 4. Route of administration |
| What excipients are used in parenteral formulations of biotech products? | Solubility enhancers Anti-adsorption and anti-aggregation agents (stops adsorption onto packaging and clumping of proteins) Buffer components Preservatives and antioxidants Osmotic agents |
| What are the microbial considerations for biotech formulations? | Sterility Viral decontamination Pyrogen removal |
| How are shelf life issues of protein based products overcome? | Proteins aren't stable in solution so formulated as a powder for reconstitution and freeze dried. |
| What routes of administration are used for biotech products? What are the issues with oral RoA? | Parenteral Oral - protein can degrade in the GI tract - no therapeutic benefit. Poor permeability of the GI wall - poor absorption. Oral vaccines Alternative routes: nasal, pulmonary, rectal, buccal, transdermal, |
| Explain the beginning of the molecular cloning process | Foreign DNA is to be inserted into a plasmid. Both the DNA and plasmid have the same restriction enzyme. RS is in middle of gene for LacZ enzyme. The RS opens up for the insertion of the foreign DNA. Plasmid also carries the ampicillin resistance gene. |
| Explain the middle of the molecular cloning process. | DNA fragment inserts into the plasmid via the sticky ends left by the RS enzyme. Sticky ends anneal, DNA ligase reattches the DNA backbones and the plasmid is now a recombinant plasmid. Plasmids are combined with a culture of living bacteria. |
| Explain the end stage of molecular cloning process | bacteria may or may not take up a plasmid. Those that do cannot produce the lacZ enzyme, DNA insert disrupts the production. The bacteria are grown on a plate with ampicillin and a reagent. Reagent changes colour if exposed to lacZ. |
| Explain the end stage of molecular cloning process cont'd. | Amipicillin kills any bacteria without the plasmid -carries the resistant gene. The bacteria with the plasmid are then isolated for growth. |
Created by:
LDM
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