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PHA112 > Microbiology 6 > Flashcards

Microbiology 6 Flashcards

1
Q

mAb upstream process

A
  • 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
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2
Q

mAb downstream process

A
  • 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
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3
Q

define purification?

A

separation of products from production mixtures/removal of unwanted components/contaminants

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4
Q

what are the different ways to purify?

A
  • sedmentation & precipitation e.g. heat, pH, organics
  • centrifugation
  • adsorption e.g. ion exchange, immuno-affinity
  • micro-filtration with specified molecular weight cut-off (MWCO)
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5
Q

why purify?

A

reduces risk of side effects while maintaining yield

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6
Q

what does yield depend on?

A
  • how many steps used

- loss of product at each step

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7
Q

what 2 processes does downstream processing involve?

A

sedimentation & precipitation

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8
Q

in sedimentation what does speed depend on?

A
  • cell size
  • density
  • mixing speed
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9
Q

what does precipitation involve?

A

lowers solute (media) solubility & causes product to fall out of solution

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10
Q

what are the various routes precipitation can be done through?

A
  • chemical
  • temp
  • pH
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11
Q

what is precipitation used in the production of?

A

recombinant DNA polymerases

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12
Q

in centrifugation, which particles move outside first?

A

denser

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13
Q

what does centrifugation require components to have?

A

different density to medium

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14
Q

what sedimentation speed depend on in centrifugation?

A
  • cell size
  • density
  • rpm
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15
Q

what is adsorption based on?

A

chromatography

passage of liquid phase through semi-solid phase

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16
Q

ion exchange

A

binds proteins based on protein charge

  • used to capture/allow passage of proteins
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17
Q

immuno-affinity

A

uses antigenic regions to bind unwanted components

  • usually targets specific contaminants
  • industrial removal of bacterial endotoxins (LPS)
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18
Q

what is sterilisation?

A

process that removes / kills everything

normally refers to bacteria & fungi

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19
Q

viruses must be removed from biologically-derived therapeutics

how so?

A

through monoclonal antibodies & plasma components

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20
Q

what may modern usage include?

A

disabling/destruction/removal of infectious proteins e.g. prions (TSE)

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21
Q

give examples of any medical product which will breach normal bodily defences against infection?

A
  1. parenteral IV administration
  2. contact with broken skin (wound dressings)
  3. contact with mucosal surfaces / internal organs
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22
Q

list the microbes in order of those most resistant to least resistant?

A
  • prions
  • spores
  • gram - bacteria
  • small non-enveloped viruses
  • fungi
  • large non-enveloped viruses
  • gram + bacteria
  • lipid enveloped viruses
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23
Q

what do prions exhibit?

A

exceptional resistance to all known sterilising agents - may even survive 18 mins at 134-138 degrees C

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24
Q

all sterilisation methods involve some risk of product damage

what can product damage reduce?

A
  • therpeutic efficiency
  • stability
  • patient acceptability
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25
Q

what are the 5 methods recognised by the European pharmacopoeia (2002)?

A
  • steam (autoclave)
  • filtration
  • dry heat (oven)
  • gas sterilisation
  • ionising radiation
26
Q

filtration …. rather than destroys microorganisms

A

removes

27
Q

what is filtration efficacy assessed by?

A

reduction in bacterial count

28
Q

what plays a role in retaining contaminants?

A
  • pore size (0.22 micrometers size used)

- composition of membrane

29
Q

MWCO filter can remove….

A

on bases of atomic mass (Da)

30
Q

what are the major uses of filtration?

A
  • heat sensitive solutions
  • biological products
  • air and other gases
  • water
31
Q

membrane filters

A

particles retained on filter surface

32
Q

depth filters

A

particles trapped in filter

33
Q

what filters can be combined?

A
  • depth (pre filter)

- membrane (sterilising)

34
Q

moist heat methods

A
  • uses hydrolytic action
  • steam at more than 120 degrees C
  • more than 1 atmosphere pressure
35
Q

dry heat methods

A
  • uses oxidative action

- temp more than 150 degrees

36
Q

what is a broad spectrum antimicrobial?

A

standard method for inactivating bio hazardous waste

37
Q

what issues are there with heat based sterilisation methods?

A

compatibility issues

38
Q

give 2 chemically reactive gases used in gas-based sterilisation methods?

A
  • ethylene oxide (CH2)2O

- formaldehyde HCHO

39
Q

what must packaging material be?

A

permeable

40
Q

what is gas sterilisation not as reliable as?

A

heat based methods

41
Q

what is gas based sterilisation generally reserved for?

A

temp sensitive items

  • reusable surgical instruments
  • medical diagnostic
  • electrical equipment
  • powders
42
Q

gas based sterilisation in broad spectrum antimicrobials

A
  • mechanism of action assumed to = alkylation of various protein FG
  • ethylene oxide = flammale, toxic and carcinogenic
43
Q

what are the 2 types of radiation used?

A

ionising- gamma rays, accelerated electrons, x-rays

non-ionising- UV light (optimum wavelength = 260nm)

44
Q

ionising radiation

A
  • facility must = heavily shielded

- can damage some material (e.g. radiolysis of water)

45
Q

non-ionising radiation

A

uv light only used for air/surface/shallow water sterilisation

  • lower energy than ionising
46
Q

what do both the ionising radiations primarily target?

A

microbial dna

47
Q

what are the various factors that can influence the outcome of such processes?

A
  • poor circulation of steam

- poor equip of maintenance/cleaning

48
Q

what are the 3 ways sterilisation can be checked?

A
  • physical indicators
  • chemical indicators
  • biological indicators
49
Q

what is the graph called for a heat sterilisation cycle?

A

temperature/pressure record chart

50
Q

where is a thermometer probe (thermocouple) located?

A

coolest part of loaded steriliser/ inserted into test packs

51
Q

what are chemical indicators based on?

A

ability to visibly alter chemical characteristics

52
Q

standardised bacterial spore preparations

A
  • non-pathogenic
  • possess good thermal resistance
  • geobacillus stearothermophillus
53
Q

process of standardising bacterial spore preparations?

A
  • placed in dummy packs located around steriliser

- after processing spores grown in nutrient medium

54
Q

in bacterial spore preparations, how can delay from incubation time be reduced?

A

using a visible indicator

pH decrease causes purple -> yellow (steam)

55
Q

on exposure to sterilisation process, what do microbial populations lose?

A

viability exponentially, independent of initial numbers

56
Q

what does sterile mean?

A

NO survivors

57
Q

what is the microbial safety index?

A

chance of single surviving organism

probability of non-sterile unit = no more than 1 in 1 million - 10^-6

58
Q

the nature of contaminant will have….

A

direct impact on success of sterilisation

59
Q

what does sterility testing assess?

A

whether sterilised product = free from microbial contamination by incubation of sample in nutrient medium

60
Q

membrane filtration in sterility testing

A
  • 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
61
Q

when using biological sterilisation indicators, what is the colour change used to check?

A

whether stability has worked or not

PHA112 (37 decks)

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