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Industrial Bioprocesses (MBIO 4520) > Final Exam > Flashcards

Final Exam Flashcards

1
Q

What does TPP stand for?

A

Target Product Profile

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2
Q

Define Target Product Profile (TPP)

A

summary of the quality characteristics of a drug product that ideally will be achieved to ensure desired quality, taking into account safety and efficacy

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3
Q

What does CQA stand for?

A

Critical Quality Attributes

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4
Q

Define Critical Quality Attributes (CQA)

A

a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality (safety parameters)

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5
Q

What are the basis for initial CQA acceptable ranges?

A
  • Specific characteristics of the product mAb, on a case-by-case basis.
  • Prior knowledge (eg. clinical studies for similar mAbs/therapeutic indications).
  • In vitro studies.
  • In vivo animal studies.
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6
Q

Define technical baseline

A

a “living” reference document(s) with which to measure progress of the project over its entirety

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7
Q

What are the 7 measures in the technical baseline?

A
  • net present value (NPV)
  • project budget
  • project strategies
  • project resources requirement
  • project milestones & timeline goals
  • project baseline
  • project plan
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8
Q

Define net present value (NPV)

A

the current value of the product relative to project status, cost-to-develop, manufacture and license, and estimated future worth baed on market projections

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9
Q

Define project budget

A

projected fixed costs, variable costs, yearly costs, cost breakdowns to understand spending patterns etc. determines project strategies depending on available cash

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10
Q

Define project strategies

A

determines technical plan manufacturing plan, regulatory plan, marketing plan, product lifecycle plan

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11
Q

Define project resources requirement

A

technical expertise, management, personnel, R&D/PD facility, manufacturing facility & equipment

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12
Q

Define project milestones & time goals

A

a measure of progress/success

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13
Q

Define product baseline

A

TPP, process consistency, CQA updates, product yield & efficacy, wuality, shelf life

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14
Q

Define project plan

A

a map of how the process will be developed to make the product (eg. GANNT chart)

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15
Q

Define quality by design

A

systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management

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16
Q

What are ICH guidances?

A

blueprints for greater worldwide harmonization to ensure that safe, effective and high quality medicines are developed and registered in the most resource-efficient manner
(Canada, USA, EU, Japan, Brazil, Korea, Singapore, China)

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17
Q

What are a couple key and critical parameters for seed trains?

A
  • temp. (key)
  • washing technique (critical)
  • pH (critical)
  • DO (critical)
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18
Q

What are some critical key process control parameters for bioreactor N performance and scale-up?

A
  • temperature (key)
  • [glucose] (critical)
  • mixing speed of additives (key)
  • raw material qualification + supply (critical)
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19
Q

What are some typical performance ranges for consistent bioreactor scale-up and mAb production?

A
  • [lactic acid] (critical)
  • osmolarity (key)
  • cell (%) viability (critical)
  • mAb productivity (key)
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20
Q

What is the flow in the development of optimal process steps under QbD using DOE statistical analysis?

A

product profile → CQAs → design space → control strategy → continual improvement
(risk assessment done in between each step)

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21
Q

Define design space (proven acceptable range)

A

parameter design and interactions (use DOE statistical analysis) - the model space which visualizes the design margin and edge of failure limits

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22
Q

Define DOE

A

design of experiments

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23
Q

Define operating range

A

the range within which product manufactured is within specifications for therapeutic use

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24
Q

Define operating settings

A

the CQA-based parameter settings used in operation, centred within the operating range

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25
Q

Define control strategy

A

tolerance design (DOE statistical analysis) leading to qualified process control

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26
Q

When cell viability is low, how do you improve existing culture media?

A

add additive to get a higher level of product

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27
Q

What are the purposes of small (PD) scale and at-scale odes development and optimization? (3 things)

A
  • characterize, optimize, and troubleshoot processes
  • account for differences between process scales in order to predict performance
  • tools to demonstrate control and to validate a process
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28
Q

What are the specific reasons for a small-scale (lab) model? (5 things)

A
  • cost + time
  • product representative of at-scale can be produced for studies when small-scale & at-scale models calibrated
  • characterization control (on-the-go) during process development studies
  • increase process knowledge and understanding, strategic direction, and prediction accuracy
  • baseline for developing the at-scale model
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29
Q

What are the specific reasons for at-scale (manufacturing) model?

A
  • understand process control in plan (eg. design margins, process centering, specifications, acceptance criteria)
  • investigating and understanding out-of-specification (OOS) events and process deviations
  • predict effects of proposed changes (different levels) for completing OOS investigation and process improvement
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30
Q

What are the scale-up criteria?

A
  • geometrical similarity
  • mass transfer coefficient
  • power per unit liquid volume
  • impeller tip velocity
  • impeller Reynolds number
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31
Q

What is the geometrical similarity criteria for scale up?

A
  • if not preserved it makes scale-up more complex

- need to follow height to depth ratio to be followed because it is very specific

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32
Q

What are the typical modality choices for downstream processing of mAb in a 2000L process?

A
  • Have bioreactor and harvest it.
  • Then we need to capture protein A.
  • Virus inactivation by low pH.
  • Use cation exchange chromatography, adjust pH.
  • Can carry out HIC and MM (HIC removed high molecular weight compounds and DNA removed, MM removes DNA and aggregates)
  • Then do virus filtration.
  • After virus inactivation could instead do DF if there is precipitate and then do the other chromatography.
  • There are 2 paths that can be followed.
  • Make sure there is no viral residues and get highly purified product.
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33
Q

What is important to note about counting mAb resins for mini-columns?

A
  • differnet resins have different binding capacities.
  • binding also dependent on loading rate (affects residence time)
  • residence time effects breakthrough time (the time a mAb flows through without binding)
  • study resumed used to choose resin & determine best residence time to maximize binding capacity to then create labs scale models to predict/analyze performance
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34
Q

What are some of the keys and critical parameters affecting performance/scale-up of mAb capture by protein A?

A
  • cost of protein A resin/column (key)
  • loading flow rate (critical)
  • RT (critical)
  • clean-in-place capability (key)
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35
Q

What is the difference between key and critical parameters?

A

key is easily controlled, critical is not easily controlled

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36
Q

What are some of the parameters affecting performance and scale-up of enveloped virus inactivation? Which are key and critical?

A
  • inactivation time (key)
  • temperature (key)
  • pH (critical)
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37
Q

What are some of the manufacturing regulatory and strategic considerations of facility design?

A

air flow directions etc.

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38
Q

What are the 4 things does titre impact when scale-up is beyond facility-design-limits?

A
  • size, type and cost of columns
  • # cycles required/step (total operating times, volumes of product)
  • volume/number of tanks (required for buffer and prep and intermediate product increases with mAb load)
  • choice of manufacturing location - might be better to contract out
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39
Q

How is maximizing a plant done for a commercial scale?

A

staggered starts with large bioreactors to produce tons of mAb/year - works most efficiently with single use equipment

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40
Q

Define distilled with regards to beer production

A

contain higher alcohol volume - it concentrates alcohol by separating water. (non-distilled has low alcohol concentration)

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41
Q

Define beer

A

alcohol-containing beverage resulting from the fermentation of sugars to ethanol by yeasts

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42
Q

What are the 4 key components of beer?

A

1) cereals/grains
2) hops
3) yeast
4) water

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43
Q

Define hops

A

plant??????

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44
Q

What effect does adding hops at different steps in beer production have?

A

varies the bitterness

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45
Q

What is the purpose of cereals/grains in beer?

A

source of fermentable carbs and sugars and nitrogen for yeasts

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46
Q

What does the different strains of yeasts do for beer production?

A

results in different types of beer

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47
Q

What are the 5 steps in beer production?

A

1) malting of barley
2) machine and wort preparation
3) fermentation
4) maturation & stabilization (post fermentation)
5) clarification + bottling

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48
Q

What happens in the first step of beer production - malting of barley?

A

breakdown of polysaccharides

-varying conditions produces beers with different colours and flavours

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49
Q

Why is malting necessary in beer production?

A

allows husks to be cracked easily. Grain looks like – outer husk and then inside there is starchy endosperm. We want to get into the starchy endosperm

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50
Q

What are the 4 steps in the malting process of beer production?

A

1) Soaking/steeping in water for 2 days to increase moisture content/prevent embryo damage
2) Partial germination
3) Malt kilning to minimize enzyme denaturation, develops flavour and colour
4) Milling (grinding to make grist)

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51
Q

Define abrasion

A

controlled damage of the husk before steeping to improve access of water and additives

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52
Q

What happens in the second step mashing and wort preparation in beer production?

A
  • produces aqueous fermentation medium - wort
  • hops added
  • sterilized by boiling
  • wort gets fermented
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53
Q

What is the carbohydrate in barley?

A

amylose

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54
Q

What happens in wort boiling in beer production?

A
  • makes more bitter acids
  • reduces bacterial load - sterilize
  • concentrate wort
  • terminate enzyme activity
  • precipitate unwanted proteins
  • remove compounds that impair flavour
  • after boiling it is clarified and cooled for fermentation
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55
Q

What happens in fermentation in beer production?

A
  • inoculated wort undergoes an alcoholic fermentation to produce ethanol, CO2 and minor metabolites that contribute to flavour and aroma
  • uses S. cerevisiae
  • after treatments are conducted to mature or condition the new beer to make it ready for consumption, which may take from one to several weeks
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56
Q

What are some additional types of adjuvants added into the fermentation process?

A
  • oxygen pre-sparge
  • bacteria
  • herbs & spices
  • chocolate
  • fruits
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57
Q

What does the amount of each beer end product depend on?

A
  • yeast strain

- fermentation conditions (temp., oxygen, C:N ratio, duration of fermentation + maturation)

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58
Q

What are the 2 things that contribute to aroma?

A
  • strains with high β-glycosidase activity

- volatile diketones also contribute to aroma

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59
Q

What are the advantages of cylindroconical vessels for fermentation?

A
  • Improved fermentation rates/control compared to a simple tank design.
  • Used to produce a range of beers.
  • Relatively low construction costs.
  • Relatively simple supporting piping etc., and footprint.
  • Large fermentation capacity.
  • Low running costs.
  • Easy CO2 collection from the top of the fermenter and yeast removal from the conical base, where yeast collects at the end of the fermentation.
  • Beer losses reduced to a minimum.
  • Efficient temperature control-got cooking jacket.
  • Consistent product quality.
  • Easy to clean using modern cleaning-in-place (CIP) systems.
  • Same vessels may also be used for beer maturation following yeast removal.
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60
Q

What type of beer does Saccharomyces cerevisiae produce? What is the fermentation of it? What is the flocculation?

A
  • ale
  • top fermenting
  • flotational flocculation
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61
Q

What type of beer does Saccharomyces pastorianus produce? What is the fermentation of it? What is the flocculation?

A
  • lager
  • bottom fermenting
  • sedimentary flocculation
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62
Q

What happens in the maturation & stabilization stage of beer production?

A
  • beer rests to release volatile components (maturation)

- add finings, sterilizing can be done before or after bottling/cold or hot (stabilization)

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63
Q

What happens in the clarification and bottling step of beer production?

A
  • filtration to give clear quality, ready for bottling, add finings to reduce haze (clarification)
  • bottling
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64
Q

When are open fermentation tanks used in beer production?

A

to ferment stouts and ales

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65
Q

What are the 6 variables in beer fermentation?

A
  • yeast strain (ale vs. lager)
  • pH
  • temperature (ale vs. lager)
  • oxygen input
  • wort nutritional status (composition)
  • yeast-wort contact (mixing)
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66
Q

Why can’t S. pastorianus be used in lager yeasts?

A

does not sporulate, meiotic recombination

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67
Q

Why is impeller use rare in beer production?

A
  • releases CO2
  • mixing damages yeast cells (apparently, but is a myth)
  • results in more consistent product
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68
Q

Why is gravity an important measurement of sugar content I+ ethanol production?

A
  • gravity/density relative to water in wort is determined by sugar content
  • decline in sugar content and presence of ethanol formation reduces gravity of wort
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69
Q

What is the magic technique used in very high gravity brewing/fermentation?

A

yeast extract, pro oxygenate yeasts prior to pitching and optimize amount of yeast cells pitched into the fermenter - leads to better alcohol tolerance

PITCHING = INNOCULATING

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70
Q

What are the differences between conventional and very high gravity fermentation?

A
  • final concentration is nearly tripled in VHG
  • 50% higher brewing capacity in VHG
  • relatively improved flavour stability in VHG
  • higher enzyme activity in VHG due to lower starch:H2O
  • spoilage bacteria reduced in VHG due to higher osmotic conditions and ethanol
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71
Q

What are the characteristics affecting beer spoilage?

A
  • Raw materials.
  • pH.
  • Ethanol content.
  • Type of ingredients (e.g., wort is rich in nutrients & therefore very susceptible).
  • Oxygen levels at various stages.
  • Level of sanitation in the brewing environment.
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72
Q

When can lactic acid bacteria spoil beer production? What effect do they have?

A
  • any steps

- flavour and appearance defects

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73
Q

When can Pediococcus sp. spoil beer production? What effect do they have?

A
  • fermentation

- produces diacetyl - undesireable flavour

74
Q

When can acetic acid bacteria spoil beer production? What effect do they have?

A
  • fermentation and post-fermentation

- convert ethanol to acetic acid when beer exposed to air

75
Q

When can Enterobacteriaceae spoil beer production? What effect do they have?

A
  • wort prior to fermentation

- spoils wort

76
Q

When can wild yeasts spoil beer production? What effect do they have?

A
  • throughout fermentation

- alter flavour and ferment beer beyond endpoint

77
Q

When can mold and other fungi (that produce mycotoxins) spoil beer production? What effect do they have?

A
  • barley prior to malting

- affect malting, mycotoxins can hamper fermentation, cause gushing/sudden CO2 release from bottled beer

78
Q

Describe Autochthonous fermentations

A
  • fermentation by brewery-resident yeasts and bacteria introduced into the cooling wort during overnight exposure in a shallow open vessel known as a “coolship”
  • succession of communities
  • fermented and matured
79
Q

What is the succession of communities in autochthonous fermentations?

A
  • Month 1-2: Enterobacteriaceae & non-Saccharomyces yeasts  aromas develop.-Month 2-6: Lactic acid bacteria & Saccharomyces sp. dominate the alcoholic fermentation.
  • Months 6+: Brettanomyces bruxellensis dominates fermentation and maturation.
80
Q

What is an example of a beer made with autochthonous fermentation?

A

lambic beers of belgium

81
Q

What are the traditional African beers that are partially autochthonous like?

A
  • consumed in femrenting state
  • back-slopping of yeasts to start batch (yeasts from a previous fermentation)
  • typically S. cerevisiae
82
Q

Define antibiotics

A

against life

83
Q

What idd Paul Vuilemin term antibiotics?

A

antibiosis - life used to destroy life

84
Q

Who discovered penicillin?

A

Alexander Fleming

85
Q

What id dCecil George Paine do with regards to penicillin?

A

treatment of infants and later adults with conjunctivitis/eye infections

86
Q

How do you test activity spectrum of antibiotics?

A
  • isolation done by spread plate, overlay with indication organisms to see zones of clearing
  • test antibiotic spectrum by streaking antibiotic producer at bottom of plate and then cross streak lines of other organisms to see growth .
87
Q

What were the achievements of the Oxford group?

A

characterized penicillin and developed a protocol to produce it in pure form - separation of penicillin from mold (critical set - extraction into organic layer then back to aqueous then freeze dry)

  • trials with mice
  • clinical trials
88
Q

How does penicillin relate to WWII?

A

used to treat early infection to soldiers in the field (diphtheria, gangrene, pneumonia, syphilis, and tuberculosis)

89
Q

What was British penicillin?

A

Penicillin F

90
Q

What was US penicillin?

A

penicillin G

91
Q

What is the mode of action of penicillin?

A

attacks nam-nag cell wall synthesis

92
Q

What 2 penicillins are produced by Penicillium chrysogenum?

A

G and V

93
Q

How are different derivatives of penicillin produced?

A

β-lactam ring fused with a thiazolidine ring to form the 6-aminopenicillanic acid (6-APA) - L-a-aminoadipate, L-cys, and L-val

94
Q

What is the difference between penicillin V and G administration?

A
  • C is oral

- G has to be injected

95
Q

What are the 4 different types of penicillin?

A
  • natural
  • biosynthetic
  • semi-synthetic
  • synthetic
96
Q

Define natural penicillin

A

produced by microorganism

97
Q

Define biosynthetic penicillin

A

chemically modified natural agents
-precursor such as phenyl acetic acid or phenoxy acetic acid is added to the culture to drive the synthesis of a particular type

98
Q

Define semi-synthetic penicillin

A

R group is stripped from a natural penicillin and another R group is added synthetically

99
Q

Define synthetic penicillin

A

chemically designed in the lab

100
Q

3 reasons to produce different forms of penicillin

A
  • resistance to penicillinase (β-lactamase, produced by some bacteria)
  • increased pH tolerance
  • increased spectrum of activity
101
Q

What are some of the downstream processing of penicillin?

A
  • For parenteral (injection) use, the antibiotic is packed in sterile vials as a powder or suspension.
  • For oral use, it is tableted usually now with a film coating.
  • Quality tests (ex: for purity, potency, stability, half-life) are performed on a specified number of random samples of the finished product, similarly to all pharmaceutical products.
  • Testing must satisfy fully all regulatory requirements before being released for sale
102
Q

What are some ways to improve/engineer strains/process for penicillin?

A
  • Further understand regulatory pathways in P. chrysogenum to look for strategies to shift metabolism into penicillin production at higher levels.
  • Over expressing the enzymes involved (increase gene copies).
  • Improved oxygen mass transfer (Bioreactor design improvement) as many steps in the biosynthesis are oxygen dependent.
  • Re-engineer the biosynthetic pathway into a heterologous “Super-host” which is easier to grow to high density in fermentations and in which one could “plug-and-play” variations of the pathway to create second generation antibiotics. E.g., Streptomyces spp., Saccharomyces cerevisiae are good candidates for this approach.
103
Q

What are stem cells?

A

undifferentiated progenitor cells that regardless of their source are capable of diving and renewing themselves for long periods, are unspecialized, and give rise to specialized cell types

104
Q

What is stem cell therapy?

A
  • treat or prevent a disease or condition.
  • key tool in regenerative medicine.
  • in treatment stem cells are expanded and then induced to differentiate into functional cell types
105
Q

What are the 4 sources of stem cells?

A
  • embryonic tissues
  • fetal tissues
  • umbilical cord blood
  • adult tissues (bone marrow, blood, adipose tissue)
106
Q

Define totipotent stem cells

A

ability of single cells to produce all known cell types (embryonic stem cells)

107
Q

Define pluripotent stem cells

A

gives rise to most but not all tissues (different lineages)

108
Q

What are the 3 types of pluripotent stem cells?

A
  • endoderm (stomach, GI tract)
  • mesoderm (muscle, bone, blood)
  • ectoderm (epidermal tissue, nervous system)
109
Q

Define multipotent stem cells

A

generate more than 2 lineages

110
Q

Define unipotent stem cell

A

give rise to only one lineage

111
Q

In vivo stem cell proliferation and cellular differentiation, what are the conditions required?

A

high cell density, enhanced cell-cell interaction, cell-matrix interaction and presence of various differentiation factors

112
Q

Describe conditions for In vitro stem cell proliferation and cellular differentiation

A
  • differentiation may delimited to proliferate stem cells

- inhibit cytodifferentiation to maximize proliferation of stem cells

113
Q

What are the 3 parameters controlling differentiation in vivo & in vitro simulation?

A
  • paracrine factors
  • homocrine factors
  • histotypic culture
114
Q

What are the paracrine factor parameters controlling differentiation in vivo & in vitro simulation?

A

proteins that diffuse over small spaces to influence changes in a neighbouring cell that may be of a different type

115
Q

What are the homocrine factor parameters controlling differentiation in vivo & in vitro simulation?

A

proeitns that mediate cell-cell interaction of closely located cells of the same type

116
Q

What are the histotypic culture parameters controlling differentiation in vivo & in vitro simulation?

A

growth & propagation of one cell line is done in engineered three-dimensional matrix

117
Q

How is pluripotent stem cells usually grown?

A
  • in a 2D monolayer
  • add micro carriers when they aggregate
  • difficult to control aggregate size and they clump together so it is hard to visualize so that you use microocapsules to prevent aggregations?
118
Q

What did the stem cells and conation open the door for?

A

therapeutic cloning to produce embryonic stem cells (from blastocysts) that are genetically identical (eg. histo-compatable) to the patient donor of the nuclei - can be used to treat degenerative disease

119
Q

Where are adult stem cells found?

A

found in blood, cornea, bone marrow, dental pulp, brain, skeletal muscle, skin, liver, pancreas, and gastrointestinal tract

120
Q

Where do adult stem cells reside?

A

in specific areas of each tissue and are thought to remain inactive until they are activated by disease or injury to the tissue

121
Q

Describe plasticity with regards to adult stem cells?

A

adult stem cells from one tissue can give rise to cell types of a completely different tissue

122
Q

What are the ways to minimize the development of neoplastic cells from iPSC?

A
  • search for a less carcinogenic gene set that is necessary and sufficient for reprograming adult cells into iPSC
  • minimize the number of genes required for reprogramming and search for non-genetic factors to optimize the function of the reduced gene set
  • develop a method to eliminate the exogenous DNA from the host genome after reprogramming
  • develop a delivery protocol for non-integrated transient genetic constructs (in development)
  • use recombinant proteins instead to reprogram the cells (in development)
123
Q

What are the benefits o human iPSC?

A
  • can be generated from humans including patients with disease
  • mature cells including nerve, heart, brain, and liver cells can be derived from these iPSC
  • allows scientists to study disease mechanisms and can be a source of cells for developing regenerative medicine therapeutics
124
Q

What is the difference between human iPSC and embryonic cells?

A

iPSCs bypass the ethical issues raised by using human embryonic cells to produce stem cells but have restricted proliferation potential compared to embryonic stem cells

125
Q

What are some current and future cell-based therapies using stem cells?

A
  • bone marrow transplant (replace stem cells after chemo)
  • management of acute graft-vs-host disease in children
  • treat blood/immunological diseases
  • blindness/vision impairment
  • brain/spinal cord injury (regrow neurons)
  • organogenesis
  • restore pancreatic insulin production
  • wound healing
  • regenerative medicine
126
Q

What are the 4 issues in stem cell research?

A
  1. Purpose of embryo creation.
  2. Viability of embryos.
  3. Embryo destruction and relief of human pain.
  4. Consent of donors.
127
Q

What are the guiding principles for hPSC research by CHIR?

A
  • The research should have potential health benefits.
  • Donor should be provided with full information regarding the research and free and informed consent should be provided by the participants.
  • Privacy and confidentiality of the participants should be respected.
  • No payment for participants regarding collecting tissues or any other reproductive material.
  • Embryos should not be created for research purposed only.
  • Human dignity, cultural and spiritual integrity should be respected all through the research.
128
Q

What are the types of hPSC research that conformed with the guidelines?

A
  • research that studies human embryonic stem cells derived from human embryos(reproductive purposes, no commercial transaction)
  • research that studies human stem cell from the umbilical cord and placenta (free + informed consent)
  • research that studies human embryonic germ cell derived from human fetal tissue or amniotic fluid (pregnant women decision)
  • research that studies human stem cell from human somatic tissues (competent person, incompetent person, dead body)
129
Q

What are the types of hPSC research that would not conform with CIHR guidelines?

A
  • create human embryo to derive stem cells

- research involving cloning

130
Q

Describe how stem cell therapy is done

A
  • Cells grown on microcarriers in a bioreactor.
  • Human induced pluripotent stem cells hiPSC.
  • Induce the stem cells to make them turn into pluripotent stem cells and want them under pluripotent conditions ready for differentiation.
  • Delivery has to be very careful so no change in cell characteristic.
  • These are autologous from the same patent so no rejection.
131
Q

What are the challenges/requirements for mass production of hPSCs?

A
  • amounts required are high
  • developing well-defined and controlled hPSC expansion & differentiation processes - need to comply with regulatory requirements, controlling differentiation: minimizing differentiation during cell expansion and control of the complex interacting / overlapping parameters required to obtain differentiation to defined progenitor cell lines after expansion
132
Q

How is GMP cell banking done for an iPSC product?

A
  • cell therapies developed from Embryonic Stem Cells, or a tissue biopsy or blood collection
  • allogeneic or Autologous stem cells are expanded from starting material (can be allogeneic ESC or aautologous/allogeneic iPSC)
  • cell bank testing for identity, potency, adventitious microorganisms, endogenous and adventitious viruses, TSE, retroviruses and other viruses is required
  • after cell exemption and differentiation - differentiated progenitor cells (product) can be cryopreserved (for future use) or used immediately as allographs in order to regenerate damaged tissue
133
Q

What are the reasons 2D cultivation of stem cells is not popular?

A
  • paracrine factor diffusion is an issue
  • difficult to monitor parameters as well
  • plate-culture processes remain cost, space, and labour intensive, and are not amenable to tight monitoring control of key and critical parameters
134
Q

What is the advantage of the 2D multi-plate single-use culture system with pH, DO, and temp control?

A

this approach mimics the 2D plates in which the cells are first cultured but allows scale-up

135
Q

Why is 3D (stirred tank bioreactor) cultivation of stem cells the best approach?

A
  • aggregates supply the micro-environment for cells while the culture medium supplies a consistent (well mixed) source of nutrients
  • process control, sampling and analyses are easier to accomplish than in a 2D system
  • cell immobilization on micro-carrier beads or within gel droplets.
  • rowing the cells under conditions in which they form micro-aggregates
136
Q

What are some key developments in culturing hPSC in stirred tank bioreactors that optimize parameters for re aggregation of cells or attachment to micro-carrier after culture passage and as cell density increases?

A
  • Depends on culture medium.
  • Micro-carrier (use with cells that are particularly sensitive to shear stress).
  • Inoculum density.
  • Mixing speed.
  • Feeding strategy.
  • Control DO at ~ 30%.
137
Q

What is the most used culture method for stem cells?

A

repeated batch

138
Q

What are the issues of perfusion of stem cells?

A

while it has the highest cell yields and highest contamination risk it has limited applications

139
Q

What are the biggest challenges with regards to commercial stem cell processes?

A
  • Aggregate size control.
  • Potential for cell differentiation.
  • Monitoring pluripotency during a run
140
Q

What is some stem cell therapy nearing therapeutic use?

A

treat osteoarthritis/trauma-induced cartilage damage

  • skin Fibroblasts used to derive iPSC for this treatment
  • the current approach is best suited to areas of minor cartilage damage
  • a major limitation at present is repairing large areas of cartilage damage because of the need to reshape the cartilage structure in vivo
141
Q

What are organdie cultures?

A
  • multi-cellular tissue proxy for natural organs grown from stem cells
  • serve as models for understanding disease and for evaluation and development of associated cell therapies
  • the models are proving valuable in disease understanding and as bridging data between in-vitro (2D cultures) and in vivo analytical results
142
Q

What is multiple sclerosis?

A

autoimmune disease of the central nervous system

143
Q

What are the current multiple sclerosis treatments?

A
  • Interferon beta (suppresses immune attacks)

- High doses of adrenocortical steroids

144
Q

What happened the stem cell therapy trials using mice with MS?

A
  • turn human stem cells into neural precursor cells
  • test effect of implanting neural precursor cells in spinal cord of mice with MS
  • hNPC’s were not detected past 8 days post transplantation
  • recovery remained
  • reduced inflammation and reduced severity of demyelination
145
Q

How does phage therapy differ from broad-spectrum antibiotics?

A

don’t wipe out beneficial bacterial flora in the intestinal tract

146
Q

Why was there a resurgence in phage therapy?

A

due to the rise of antibiotic-resistant bacteria

147
Q

What are the 2 of E. coli (coliphage) phage from the family mycoviridae?

A
  • T4

- lambda

148
Q

What is of E. coli (coliphage) phage from the family inoviridae?

A

filamentous phage

149
Q

What are lytic phage best for?

A

anti-bavterial therapy (reduce lateral genetic transfer)

150
Q

What is the issue with phage that impacts purification?

A

phage are very small in size

151
Q

What can the phage genome be like?

A
  • DNA, RNA
  • single-stranded, double stranded
  • circular, linear
152
Q

In the mechanism of action of lytic bacteriophage, what causes lysis?

A
  • amurins – inhibit synthesis of peptidoglycan to destroy cell wall.
  • two-protein system (Holins & Endolysins
153
Q

How did Hershey & chase confirmed the means by which T2-phage infect bacteria?

A
  • used radio labeled phage to trace distribution
  • mix bacteria and phage holding bacteria within latent period before cells lyse
  • spin a few mins in blender
  • centrifuge to pellet bacteria
154
Q

How is bacteriophage produced in culture?

A
  • seed culture train
  • inocculate production fermenter to grow to exponential phase
  • infect exponential phase culture with phage (MOI constant - bacteria:phage ratio)
  • monitor phage titre indirectly based on DNA levels in supernatant
  • add EDTA to prevent DNA breakdown (easier to remove DNA)
  • depth filtration to remove E. coli cells, cell debris etc. & recover culture supernatant (contains phage)
155
Q

What is used to capture and purify phage>

A

DEAE (AEX) Disk monolith column Chromatography

156
Q

Why is DEAE (AEX) Disk monolith column Chromatography used to capture and purify phage?

A
  • Preferred for virus purification (flow characteristics).
  • Remove E. coli (host) DNA.
  • High binding capacity.
  • Great Scalability: Increase disks up to total volume 8L; flow rate independent.
  • Single-use available.
157
Q

What are the polishing steps of phage capture and purification?

A

remove residual DNA & HCP, endotoxins etc.

158
Q

What in the human immune system can inhibit phage effectiveness?

A

Anti-Phage Neutralizing antibodies

159
Q

What are the ways to deal with reduced efficacy of phage therapeutics owing to anti-phage antibodies?

A
  1. Repeat phage administration.
  2. Increase the dosage of phage.
  3. Use different phage, because immune response differs from one phage to another.
  4. Use a cocktail of phage (Increases anti-microbial activity, Decrease the risk of phage-resistant bacteria, Up to 30 phage strains have been used (in USSR) in purulent infections involving up to 5 types of bacteria (regulatory headache))
160
Q

How does the phage and immune system work in synergy?

A
  • bacteria invade human
  • bacteria start multiplying
  • bacteria form biofilm
  • admin of phage
  • phage breakdown biofilm/reduce bacteria number
  • innate effector cells Geta access to bacteria
  • elimination of bacteria by effector cells + phage
161
Q

What is the phage resistance in bacteria like?

A

bacteria can evolve to resist phage but phage evolve at a faster rate than biochemists can modify or create new antibiotics

162
Q

What are the challenges associated with phage therapy and solutions?

A
  • lack of knowledge
  • half-life of phage in blood is low
  • GI tract is anaerobic limits host proliferation
  • low pH & proteolytic activity limits oral admin
  • phage resistance of bacteria can develop
163
Q

What are some novel variations on phage therapy in development?

A
  • prophylactic phage therapy - oral admin
  • reduce pathogen levels in poultry
  • general disinfectant
164
Q

What are therapies in development with regards to phage?

A

personalized phage therapy for patients with very tough infection

165
Q

What are some obstacles of commercializing phage therapy?

A
  • cannot patent natural phage

- no established regulatory pathway for drug approval

166
Q

What is the present day status of phage therapy?

A
  • not approves for use in humans yet

- approved for use in food safety and agriculture

167
Q

What are T cells?

A
  • lymphocytes of the adaptive IS that “roam” the human circulation system detecting “foreign or abnormal” components
  • display MHC on surface
  • bind to foreign MHC Ag via TCR
  • release cytokines to produce Th cells and killer T cells and to attract macrophages and NK cells
168
Q

What are T cells limitations to cancer?

A
  • self-MHC present on cancer and normal cells

- T-cells don’t always have the correct receptors to recognize additional cancer-specific, cell-surface antigens

169
Q

What are chimeric antigen receptor (CAR) T cells?

A

genetically engineered to express receptors for cancer-specific antigens and target the cancer cells without engaging the TCR: MHC (cell-type), antigenic mechanism

  • CAR T cells have a genetically engineered receptor and binds to the B cell antibodies that can bind the cancer cells causes the T cells and stuff to get activated to kill the cancer cells
  • not MHC dependent so don’t need MHC - recognize cancer cells directly causing T cells to activate and kill cancer cells
170
Q

What is CAR T-cell immunotherapy?

A
  • infusion of cultured, genetically modified autologous or allogeneic T cells
  • Genetic modification (expression of a chimeric antigen receptor (CAR) on the T-cell surface)
171
Q

Compare the structure of natural TCR vs. CAR in T-cells

A
  • TCRs → Natural T-Cell receptor - binds peptides covalently linked to MHC on the surface of tumor cells
  • CAR fusion proteins have 3 segments → extracellular specific antigen-binding protein, typically a scFv from a mAb, a trans membrane moiety-goes through membrane, intracellular signaling peptide domains
172
Q

What is the difference of how TCR and CAR bind cancer cells?

A

CAR T cells do not require costimulation that normal T cells require and know cancer cells can hide their Ag

173
Q

Compare CAR- and TCR-engineered T-cell characteristics

A
  • CAR T cells have precise targeting of specific cancer antigen only
  • CAR T cells have MHC-independent recognition of targets
  • TCR had low-avidity unless engineered where CAR has controllable binding avidity
  • TCR has lifeline persistence where CAR has at least a decade
  • Both kill tumour cells
  • CRS/target normal cells can occur in TCR - CRS is more severe in CAR due to sighing avidity
174
Q

What is avidity?

A

binding strength measurement

175
Q

What is cytokine release syndrome (CRS) associated with CAR T-cell therapy?

A

occurs with T cells targeting CD19 or BCMA - it is when cytokines released increase permeability to soluble mediators and permit increased trafficking of lymphocytes to CNS
-vascular permeability increases, causes neurotoxicity

176
Q

What is the rationale of creating universal CAR T-cell therapies?

A
  • Using allogeneic donors or “universal” T-cells.
  • Simplified manufacturing.
  • Increased drug availability + lower cost.
  • “Off-the-shelf” CAR T-cell therapies
177
Q

What is the rationale of creating universal CAR T-cell therapies?

A
  • Using allogeneic donors or “universal” T-cells.
  • Simplified manufacturing.
  • Increased drug availability + lower cost.
  • “Off-the-shelf” CAR T-cell therapies
178
Q

What is the potential of creating a universal CAR T-cell therapy?

A
  • “Early Days”, this technology is in its infancy.
  • Given rapid progress in the field it is likely universal CAR T-cells will become widely used.
  • Unclear whether or not this approach will yield stand-alone therapy or be used as a bridge (quick response, lower cancer load) for successful therapies based on stem cell transplant or autologous CAR-T cells
179
Q

What are some examples of diseases tested using CAR T-cell therapy?

A
  • solid tunours

- multiple myeloma (CD19)

180
Q

What is the benefit of using CAR T-cell therapy along with chemo?

A

Chemo attacks B cells and reduces competition that improves the ability of the CAR T-cells to expand and multiply

181
Q

What are the challenges of CAR T-cell product commercialization?

A
  • Understanding long-term safety.
  • PRICE: the very high price raises questions.
  • Anticipating global regulatory concerns & unfolding changes with time
182
Q

What are the opportunities of CAR T-cell product commercialization?

A

-Expanding the type of cancers treatable to include solid tumors

Industrial Bioprocesses (MBIO 4520) (4 decks)

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