Microbiology 6

Question 1

mAb upstream process

Answer 1

• 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

Question 2

mAb downstream process

Answer 2

• 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

Question 3

define purification?

Answer 3

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

Question 4

what are the different ways to purify?

Answer 4

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

Question 5

why purify?

Answer 5

reduces risk of side effects while maintaining yield

Question 6

what does yield depend on?

Answer 6

• how many steps used

- loss of product at each step

Question 7

what 2 processes does downstream processing involve?

Answer 7

sedimentation & precipitation

Question 8

in sedimentation what does speed depend on?

Answer 8

• cell size
• density
• mixing speed

Question 9

what does precipitation involve?

Answer 9

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

Question 10

what are the various routes precipitation can be done through?

Answer 10

• chemical
• temp
• pH

Question 11

what is precipitation used in the production of?

Answer 11

recombinant DNA polymerases

Question 12

in centrifugation, which particles move outside first?

Answer 12

denser

Question 13

what does centrifugation require components to have?

Answer 13

different density to medium

Question 14

what sedimentation speed depend on in centrifugation?

Answer 14

• cell size
• density
• rpm

Question 15

what is adsorption based on?

Answer 15

chromatography

passage of liquid phase through semi-solid phase

Question 16

ion exchange

Answer 16

binds proteins based on protein charge

• used to capture/allow passage of proteins

Question 17

immuno-affinity

Answer 17

uses antigenic regions to bind unwanted components

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

Question 18

what is sterilisation?

Answer 18

process that removes / kills everything

normally refers to bacteria & fungi

Question 19

viruses must be removed from biologically-derived therapeutics

how so?

Answer 19

through monoclonal antibodies & plasma components

Question 20

what may modern usage include?

Answer 20

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

Question 21

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

Answer 21

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

Question 22

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

Answer 22

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

Question 23

what do prions exhibit?

Answer 23

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

Question 24

all sterilisation methods involve some risk of product damage

what can product damage reduce?

Answer 24

• therpeutic efficiency
• stability
• patient acceptability

Question 25

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

Answer 25

- steam (autoclave) - filtration - dry heat (oven) - gas sterilisation - ionising radiation

Question 26

filtration …. rather than destroys microorganisms

Answer 26

removes

Question 27

what is filtration efficacy assessed by?

Answer 27

reduction in bacterial count

Question 28

what plays a role in retaining contaminants?

Answer 28

- pore size (0.22 micrometers size used) | - composition of membrane

Question 29

MWCO filter can remove….

Answer 29

on bases of atomic mass (Da)

Question 30

what are the major uses of filtration?

Answer 30

- heat sensitive solutions - biological products - air and other gases - water

Question 31

membrane filters

Answer 31

particles retained on filter surface

Question 32

depth filters

Answer 32

particles trapped in filter

Question 33

what filters can be combined?

Answer 33

- depth (pre filter) | - membrane (sterilising)

Question 34

moist heat methods

Answer 34

- uses hydrolytic action - steam at more than 120 degrees C - more than 1 atmosphere pressure

Question 35

dry heat methods

Answer 35

- uses oxidative action | - temp more than 150 degrees

Question 36

what is a broad spectrum antimicrobial?

Answer 36

standard method for inactivating bio hazardous waste

Question 37

what issues are there with heat based sterilisation methods?

Answer 37

compatibility issues

Question 38

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

Answer 38

- ethylene oxide (CH2)2O | - formaldehyde HCHO

Question 39

what must packaging material be?

Answer 39

permeable

Question 40

what is gas sterilisation not as reliable as?

Answer 40

heat based methods

Question 41

what is gas based sterilisation generally reserved for?

Answer 41

temp sensitive items - reusable surgical instruments - medical diagnostic - electrical equipment - powders

Question 42

gas based sterilisation in broad spectrum antimicrobials

Answer 42

- mechanism of action assumed to = alkylation of various protein FG - ethylene oxide = flammale, toxic and carcinogenic

Question 43

what are the 2 types of radiation used?

Answer 43

ionising- gamma rays, accelerated electrons, x-rays non-ionising- UV light (optimum wavelength = 260nm)

Question 44

ionising radiation

Answer 44

- facility must = heavily shielded | - can damage some material (e.g. radiolysis of water)

Question 45

non-ionising radiation

Answer 45

uv light only used for air/surface/shallow water sterilisation - lower energy than ionising

Question 46

what do both the ionising radiations primarily target?

Answer 46

microbial dna

Question 47

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

Answer 47

- poor circulation of steam | - poor equip of maintenance/cleaning

Question 48

what are the 3 ways sterilisation can be checked?

Answer 48

- physical indicators - chemical indicators - biological indicators

Question 49

what is the graph called for a heat sterilisation cycle?

Answer 49

temperature/pressure record chart

Question 50

where is a thermometer probe (thermocouple) located?

Answer 50

coolest part of loaded steriliser/ inserted into test packs

Question 51

what are chemical indicators based on?

Answer 51

ability to visibly alter chemical characteristics

Question 52

standardised bacterial spore preparations

Answer 52

- non-pathogenic - possess good thermal resistance - geobacillus stearothermophillus

Question 53

process of standardising bacterial spore preparations?

Answer 53

- placed in dummy packs located around steriliser | - after processing spores grown in nutrient medium

Question 54

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

Answer 54

using a visible indicator pH decrease causes purple -> yellow (steam)

Question 55

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

Answer 55

viability exponentially, independent of initial numbers

Question 56

what does sterile mean?

Answer 56

NO survivors

Question 57

what is the microbial safety index?

Answer 57

chance of single surviving organism | probability of non-sterile unit = no more than 1 in 1 million - 10^-6

Question 58

the nature of contaminant will have….

Answer 58

direct impact on success of sterilisation

Question 59

what does sterility testing assess?

Answer 59

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

Question 60

membrane filtration in sterility testing

Answer 60

- 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

Question 61

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

Answer 61

whether stability has worked or not