Microbiology 6 Flashcards
mAb upstream process
- vial thaw
- expansion of cells through incoulum steps
- cells further expanded in seed bioreactors before transfer to production bioreactor
- where mAb expressed into medium
- centrifugation & filtration used to harvest cell culture broth from cells and cell debris
mAb downstream process
- protein A chromatographic capture
- 2 chromatographic steps for impurity removal
- 2 orthogonal steps for viral clearance
- low pH viral inactivation after protein A chromatography and viral filtration
- ultrafiltration/defiltration to formulate and concentrate product
define purification?
separation of products from production mixtures/removal of unwanted components/contaminants
what are the different ways to purify?
- sedmentation & precipitation e.g. heat, pH, organics
- centrifugation
- adsorption e.g. ion exchange, immuno-affinity
- micro-filtration with specified molecular weight cut-off (MWCO)
why purify?
reduces risk of side effects while maintaining yield
what does yield depend on?
- how many steps used
- loss of product at each step
what 2 processes does downstream processing involve?
sedimentation & precipitation
in sedimentation what does speed depend on?
- cell size
- density
- mixing speed
what does precipitation involve?
lowers solute (media) solubility & causes product to fall out of solution
what are the various routes precipitation can be done through?
- chemical
- temp
- pH
what is precipitation used in the production of?
recombinant DNA polymerases
in centrifugation, which particles move outside first?
denser
what does centrifugation require components to have?
different density to medium
what sedimentation speed depend on in centrifugation?
- cell size
- density
- rpm
what is adsorption based on?
chromatography
passage of liquid phase through semi-solid phase
ion exchange
binds proteins based on protein charge
- used to capture/allow passage of proteins
immuno-affinity
uses antigenic regions to bind unwanted components
- usually targets specific contaminants
- industrial removal of bacterial endotoxins (LPS)
what is sterilisation?
process that removes / kills everything
normally refers to bacteria & fungi
viruses must be removed from biologically-derived therapeutics
how so?
through monoclonal antibodies & plasma components
what may modern usage include?
disabling/destruction/removal of infectious proteins e.g. prions (TSE)
give examples of any medical product which will breach normal bodily defences against infection?
- parenteral IV administration
- contact with broken skin (wound dressings)
- contact with mucosal surfaces / internal organs
list the microbes in order of those most resistant to least resistant?
- prions
- spores
- gram - bacteria
- small non-enveloped viruses
- fungi
- large non-enveloped viruses
- gram + bacteria
- lipid enveloped viruses
what do prions exhibit?
exceptional resistance to all known sterilising agents - may even survive 18 mins at 134-138 degrees C
all sterilisation methods involve some risk of product damage
what can product damage reduce?
- therpeutic efficiency
- stability
- patient acceptability
what are the 5 methods recognised by the European pharmacopoeia (2002)?
- steam (autoclave)
- filtration
- dry heat (oven)
- gas sterilisation
- ionising radiation
filtration …. rather than destroys microorganisms
removes
what is filtration efficacy assessed by?
reduction in bacterial count
what plays a role in retaining contaminants?
- pore size (0.22 micrometers size used)
- composition of membrane
MWCO filter can remove….
on bases of atomic mass (Da)
what are the major uses of filtration?
- heat sensitive solutions
- biological products
- air and other gases
- water
membrane filters
particles retained on filter surface
depth filters
particles trapped in filter
what filters can be combined?
- depth (pre filter)
- membrane (sterilising)
moist heat methods
- uses hydrolytic action
- steam at more than 120 degrees C
- more than 1 atmosphere pressure
dry heat methods
- uses oxidative action
- temp more than 150 degrees
what is a broad spectrum antimicrobial?
standard method for inactivating bio hazardous waste
what issues are there with heat based sterilisation methods?
compatibility issues
give 2 chemically reactive gases used in gas-based sterilisation methods?
- ethylene oxide (CH2)2O
- formaldehyde HCHO
what must packaging material be?
permeable
what is gas sterilisation not as reliable as?
heat based methods
what is gas based sterilisation generally reserved for?
temp sensitive items
- reusable surgical instruments
- medical diagnostic
- electrical equipment
- powders
gas based sterilisation in broad spectrum antimicrobials
- mechanism of action assumed to = alkylation of various protein FG
- ethylene oxide = flammale, toxic and carcinogenic
what are the 2 types of radiation used?
ionising- gamma rays, accelerated electrons, x-rays
non-ionising- UV light (optimum wavelength = 260nm)
ionising radiation
- facility must = heavily shielded
- can damage some material (e.g. radiolysis of water)
non-ionising radiation
uv light only used for air/surface/shallow water sterilisation
- lower energy than ionising
what do both the ionising radiations primarily target?
microbial dna
what are the various factors that can influence the outcome of such processes?
- poor circulation of steam
- poor equip of maintenance/cleaning
what are the 3 ways sterilisation can be checked?
- physical indicators
- chemical indicators
- biological indicators
what is the graph called for a heat sterilisation cycle?
temperature/pressure record chart
where is a thermometer probe (thermocouple) located?
coolest part of loaded steriliser/ inserted into test packs
what are chemical indicators based on?
ability to visibly alter chemical characteristics
standardised bacterial spore preparations
- non-pathogenic
- possess good thermal resistance
- geobacillus stearothermophillus
process of standardising bacterial spore preparations?
- placed in dummy packs located around steriliser
- after processing spores grown in nutrient medium
in bacterial spore preparations, how can delay from incubation time be reduced?
using a visible indicator
pH decrease causes purple -> yellow (steam)
on exposure to sterilisation process, what do microbial populations lose?
viability exponentially, independent of initial numbers
what does sterile mean?
NO survivors
what is the microbial safety index?
chance of single surviving organism
probability of non-sterile unit = no more than 1 in 1 million - 10^-6
the nature of contaminant will have….
direct impact on success of sterilisation
what does sterility testing assess?
whether sterilised product = free from microbial contamination by incubation of sample in nutrient medium
membrane filtration in sterility testing
- method of choice for pharmaceutical products
- microorganisms from liquid product collected on sterile filter
- filter transferred to appropriate media
- long incubation times e.g. 14 days
- visual inspection for turbidity
when using biological sterilisation indicators, what is the colour change used to check?
whether stability has worked or not
