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25 Cards in this Set
- Front
- Back
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What are some different expression systems?
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microbial
yeast insect plant mammalian hybridoma human |
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What can glycoform profile of a protein be infulenced by?
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expression system used
cell culture conditions temp growth rate many selectively purfy a particular PTM product variant |
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Where to Post translational modifications happen? Where do physicochemical changes happen?
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In the Cell-dependent on gene construct experssion system
During manufacturing process |
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What are two main types of protein changes that can happen during manufacturing? describe each
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Noncovalent and Covalent
noncovalent-denaturation, aggregation, precipitation, adsorption Covalent-Deamidation, oxidation, disulfide exchange, proteolysis |
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When can aggregation occur?
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During cell culture
during purification elution conditions during formulation during filing during storage |
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What are different mammalian cell expression systems?
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Transient expression- product gene is introduced into host cell and maintained/replicated as an extra-chromosomal unit
Stable expression-product gene must be integrated into host cell genome for stable production |
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What are different transfection methods?
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Calcium phosphate-DNA trapped by CaPi precipitate and uptaken by call and transported to nucleus
lipid-mediated + charged lipids bind to - charged DNA englufed by endocytosis electroporation- biomembrans become transiently permeable when shocked |
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What happens from T-flask to bioreactor in cell banking?
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happens when a single vial of working cell bank system is taken from storage and cells cultured in order to initiate the biosysnthsis of a batch product
1)thaw vial 2)laboratore scale starter culture 3)produciton scale starter culture 4)produciton scale bioractor |
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In cell banking viability has to be more than what percent? and how are the cell lines preserved
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more than 90%
they are preserved in liquid nitrogen. |
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As cells are frozen by degrees celcius/min what helps make intracellular freezing less likley?
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They get dehydrated the lower the temperature
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What makes cryoprotective compunds special?
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they are freely permeable across the cell membrane
DMSO they reduce the injury of cells during freezing |
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How does master cell banking work?
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one aliquot of master bank thawed and used to generate working cell bank and 1 working cell bank used before moving onto 2
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What cell line is used in mammalian cell transfection, expression and larg-scale production?
What is their equivilant? Why to they offer a PTM product? |
Chinses hamster ovary
E. Coli They provide stable an accurate glycosylation |
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What is the some problems with mammalian processing?
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contamination by viruses
have to do assays and filtrations to removed and prove they are removed during processing |
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What are the different biologics saftey testings?
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molecular biology-molecular analysis provides info on indifying cells and presence of viruses
microbiology-test for mycoplasma, bacterial and funal contamination virology- test for viral contaminents cell biology-provides info fo teh characterization of cell substrates |
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What is a clean room?
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a environment where airborne particulates are controlled through an exchange of highly filtered air
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What are different biomanufacturing processes?
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Process development and R&D (1-20L scale)
Pilot Studies(20-200L scale) GMP(>1,000L) QA/QC |
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What are some typical impurities in cell culture broth?
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cell debris
endotoxins DNA host cell proteins viruses aggregates/fragments media content(lipids) |
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what are the three different gasses in mammalian cell growth?
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air
carbon dioxide (co2) O2 |
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What are the two different mammalian cell types?
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Coninuous-grow in suspension
Noncontinuous-adherent, monolayer, finite lifespan,long doubling time |
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What are some requirements for a bioractor for animal cell culture?
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well controled environment
supply of nuterients gentle mixing gentle aeration removal of waste |
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What are parameters for medium optimization?
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control of phisiological parameters
oxygen co2 pH Nutrient concentrations |
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What are some differences between a fermentor and a bioractor?
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no baffle in bioractor
difference in impellers (mammalian is marine type) mammalian needs aeration but not too much so as not to damage cells |
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What are the different types of filtration?
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MF-microfiltration used in upstream to separate intact cells/cell debris from rest of components in feed stream size is 1-1000 micro meters
UF and TFF-used to separate protein from buffer components for buffer exchange size is 1-1000kD DF-same as UF except volum is kept constant by adding buffer-want to remove salts, change pH precedes chromatography |
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What is the differenct between tangential(cross flow) filtration and normal flow filtration?
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feed flow, pressure and filtrate all in same direction in normal flow, clogging can happen more easily
cross flow-feed stream moves paralles to membrane and pressure is perpindicular to flow clogging partially elminated |
