Final Exam Flashcards
1
Q
What does TPP stand for?
A
Target Product Profile
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
3
Q
What does CQA stand for?
A
Critical Quality Attributes
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)
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.
6
Q
Define technical baseline
A
a “living” reference document(s) with which to measure progress of the project over its entirety
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
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
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
10
Q
Define project strategies
A
determines technical plan manufacturing plan, regulatory plan, marketing plan, product lifecycle plan
11
Q
Define project resources requirement
A
technical expertise, management, personnel, R&D/PD facility, manufacturing facility & equipment
12
Q
Define project milestones & time goals
A
a measure of progress/success
13
Q
Define product baseline
A
TPP, process consistency, CQA updates, product yield & efficacy, wuality, shelf life
14
Q
Define project plan
A
a map of how the process will be developed to make the product (eg. GANNT chart)
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
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)
17
Q
What are a couple key and critical parameters for seed trains?
A
- temp. (key)
- washing technique (critical)
- pH (critical)
- DO (critical)
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)
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)
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)
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
22
Q
Define DOE
A
design of experiments
23
Q
Define operating range
A
the range within which product manufactured is within specifications for therapeutic use
24
Q
Define operating settings
A
the CQA-based parameter settings used in operation, centred within the operating range
25
Define control strategy
tolerance design (DOE statistical analysis) leading to qualified process control
26
When cell viability is low, how do you improve existing culture media?
add additive to get a higher level of product
27
What are the purposes of small (PD) scale and at-scale odes development and optimization? (3 things)
- 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
28
What are the specific reasons for a small-scale (lab) model? (5 things)
- 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
29
What are the specific reasons for at-scale (manufacturing) model?
- 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
30
What are the scale-up criteria?
- geometrical similarity
- mass transfer coefficient
- power per unit liquid volume
- impeller tip velocity
- impeller Reynolds number
31
What is the geometrical similarity criteria for scale up?
- if not preserved it makes scale-up more complex
| - need to follow height to depth ratio to be followed because it is very specific
32
What are the typical modality choices for downstream processing of mAb in a 2000L process?
* 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.
33
What is important to note about counting mAb resins for mini-columns?
- 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
34
What are some of the keys and critical parameters affecting performance/scale-up of mAb capture by protein A?
- cost of protein A resin/column (key)
- loading flow rate (critical)
- RT (critical)
- clean-in-place capability (key)
35
What is the difference between key and critical parameters?
key is easily controlled, critical is not easily controlled
36
What are some of the parameters affecting performance and scale-up of enveloped virus inactivation? Which are key and critical?
- inactivation time (key)
- temperature (key)
- pH (critical)
37
What are some of the manufacturing regulatory and strategic considerations of facility design?
air flow directions etc.
38
What are the 4 things does titre impact when scale-up is beyond facility-design-limits?
- 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
39
How is maximizing a plant done for a commercial scale?
staggered starts with large bioreactors to produce tons of mAb/year - works most efficiently with single use equipment
40
Define distilled with regards to beer production
contain higher alcohol volume - it concentrates alcohol by separating water. (non-distilled has low alcohol concentration)
41
Define beer
alcohol-containing beverage resulting from the fermentation of sugars to ethanol by yeasts
42
What are the 4 key components of beer?
1) cereals/grains
2) hops
3) yeast
4) water
43
Define hops
plant??????
44
What effect does adding hops at different steps in beer production have?
varies the bitterness
45
What is the purpose of cereals/grains in beer?
source of fermentable carbs and sugars and nitrogen for yeasts
46
What does the different strains of yeasts do for beer production?
results in different types of beer
47
What are the 5 steps in beer production?
1) malting of barley
2) machine and wort preparation
3) fermentation
4) maturation & stabilization (post fermentation)
5) clarification + bottling
48
What happens in the first step of beer production - malting of barley?
breakdown of polysaccharides
| -varying conditions produces beers with different colours and flavours
49
Why is malting necessary in beer production?
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
50
What are the 4 steps in the malting process of beer production?
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)
51
Define abrasion
controlled damage of the husk before steeping to improve access of water and additives
52
What happens in the second step mashing and wort preparation in beer production?
- produces aqueous fermentation medium - wort
- hops added
- sterilized by boiling
- wort gets fermented
53
What is the carbohydrate in barley?
amylose
54
What happens in wort boiling in beer production?
- 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
55
What happens in fermentation in beer production?
- 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
56
What are some additional types of adjuvants added into the fermentation process?
- oxygen pre-sparge
- bacteria
- herbs & spices
- chocolate
- fruits
57
What does the amount of each beer end product depend on?
- yeast strain
| - fermentation conditions (temp., oxygen, C:N ratio, duration of fermentation + maturation)
58
What are the 2 things that contribute to aroma?
- strains with high β-glycosidase activity
| - volatile diketones also contribute to aroma
59
What are the advantages of cylindroconical vessels for fermentation?
* 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.
60
What type of beer does Saccharomyces cerevisiae produce? What is the fermentation of it? What is the flocculation?
- ale
- top fermenting
- flotational flocculation
61
What type of beer does Saccharomyces pastorianus produce? What is the fermentation of it? What is the flocculation?
- lager
- bottom fermenting
- sedimentary flocculation
62
What happens in the maturation & stabilization stage of beer production?
- beer rests to release volatile components (maturation)
| - add finings, sterilizing can be done before or after bottling/cold or hot (stabilization)
63
What happens in the clarification and bottling step of beer production?
- filtration to give clear quality, ready for bottling, add finings to reduce haze (clarification)
- bottling
64
When are open fermentation tanks used in beer production?
to ferment stouts and ales
65
What are the 6 variables in beer fermentation?
- yeast strain (ale vs. lager)
- pH
- temperature (ale vs. lager)
- oxygen input
- wort nutritional status (composition)
- yeast-wort contact (mixing)
66
Why can't S. pastorianus be used in lager yeasts?
does not sporulate, meiotic recombination
67
Why is impeller use rare in beer production?
- releases CO2
- mixing damages yeast cells (apparently, but is a myth)
- results in more consistent product
68
Why is gravity an important measurement of sugar content I+ ethanol production?
- 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
69
What is the magic technique used in very high gravity brewing/fermentation?
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
70
What are the differences between conventional and very high gravity fermentation?
- 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
71
What are the characteristics affecting beer spoilage?
- 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.
72
When can lactic acid bacteria spoil beer production? What effect do they have?
- any steps
| - flavour and appearance defects
73
When can Pediococcus sp. spoil beer production? What effect do they have?
- fermentation
| - produces diacetyl - undesireable flavour
74
When can acetic acid bacteria spoil beer production? What effect do they have?
- fermentation and post-fermentation
| - convert ethanol to acetic acid when beer exposed to air
75
When can Enterobacteriaceae spoil beer production? What effect do they have?
- wort prior to fermentation
| - spoils wort
76
When can wild yeasts spoil beer production? What effect do they have?
- throughout fermentation
| - alter flavour and ferment beer beyond endpoint
77
When can mold and other fungi (that produce mycotoxins) spoil beer production? What effect do they have?
- barley prior to malting
| - affect malting, mycotoxins can hamper fermentation, cause gushing/sudden CO2 release from bottled beer
78
Describe Autochthonous fermentations
- 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
What is the succession of communities in autochthonous fermentations?
- 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
What is an example of a beer made with autochthonous fermentation?
lambic beers of belgium
81
What are the traditional African beers that are partially autochthonous like?
- consumed in femrenting state
- back-slopping of yeasts to start batch (yeasts from a previous fermentation)
- typically S. cerevisiae
82
Define antibiotics
against life
83
What idd Paul Vuilemin term antibiotics?
antibiosis - life used to destroy life
84
Who discovered penicillin?
Alexander Fleming
85
What id dCecil George Paine do with regards to penicillin?
treatment of infants and later adults with conjunctivitis/eye infections
86
How do you test activity spectrum of antibiotics?
- 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
What were the achievements of the Oxford group?
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
How does penicillin relate to WWII?
used to treat early infection to soldiers in the field (diphtheria, gangrene, pneumonia, syphilis, and tuberculosis)
89
What was British penicillin?
Penicillin F
90
What was US penicillin?
penicillin G
91
What is the mode of action of penicillin?
attacks nam-nag cell wall synthesis
92
What 2 penicillins are produced by Penicillium chrysogenum?
G and V
93
How are different derivatives of penicillin produced?
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
What is the difference between penicillin V and G administration?
- C is oral
| - G has to be injected
95
What are the 4 different types of penicillin?
- natural
- biosynthetic
- semi-synthetic
- synthetic
96
Define natural penicillin
produced by microorganism
97
Define biosynthetic penicillin
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
Define semi-synthetic penicillin
R group is stripped from a natural penicillin and another R group is added synthetically
99
Define synthetic penicillin
chemically designed in the lab
100
3 reasons to produce different forms of penicillin
- resistance to penicillinase (β-lactamase, produced by some bacteria)
- increased pH tolerance
- increased spectrum of activity
101
What are some of the downstream processing of penicillin?
* 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
What are some ways to improve/engineer strains/process for penicillin?
* 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
What are stem cells?
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
What is stem cell therapy?
- 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
What are the 4 sources of stem cells?
- embryonic tissues
- fetal tissues
- umbilical cord blood
- adult tissues (bone marrow, blood, adipose tissue)
106
Define totipotent stem cells
ability of single cells to produce all known cell types (embryonic stem cells)
107
Define pluripotent stem cells
gives rise to most but not all tissues (different lineages)
108
What are the 3 types of pluripotent stem cells?
- endoderm (stomach, GI tract)
- mesoderm (muscle, bone, blood)
- ectoderm (epidermal tissue, nervous system)
109
Define multipotent stem cells
generate more than 2 lineages
110
Define unipotent stem cell
give rise to only one lineage
111
In vivo stem cell proliferation and cellular differentiation, what are the conditions required?
high cell density, enhanced cell-cell interaction, cell-matrix interaction and presence of various differentiation factors
112
Describe conditions for In vitro stem cell proliferation and cellular differentiation
- differentiation may delimited to proliferate stem cells
| - inhibit cytodifferentiation to maximize proliferation of stem cells
113
What are the 3 parameters controlling differentiation in vivo & in vitro simulation?
- paracrine factors
- homocrine factors
- histotypic culture
114
What are the paracrine factor parameters controlling differentiation in vivo & in vitro simulation?
proteins that diffuse over small spaces to influence changes in a neighbouring cell that may be of a different type
115
What are the homocrine factor parameters controlling differentiation in vivo & in vitro simulation?
proeitns that mediate cell-cell interaction of closely located cells of the same type
116
What are the histotypic culture parameters controlling differentiation in vivo & in vitro simulation?
growth & propagation of one cell line is done in engineered three-dimensional matrix
117
How is pluripotent stem cells usually grown?
- 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
What did the stem cells and conation open the door for?
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
Where are adult stem cells found?
found in blood, cornea, bone marrow, dental pulp, brain, skeletal muscle, skin, liver, pancreas, and gastrointestinal tract
120
Where do adult stem cells reside?
in specific areas of each tissue and are thought to remain inactive until they are activated by disease or injury to the tissue
121
Describe plasticity with regards to adult stem cells?
adult stem cells from one tissue can give rise to cell types of a completely different tissue
122
What are the ways to minimize the development of neoplastic cells from iPSC?
- 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
What are the benefits o human iPSC?
- 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
What is the difference between human iPSC and embryonic cells?
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
What are some current and future cell-based therapies using stem cells?
- 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
What are the 4 issues in stem cell research?
1. Purpose of embryo creation.
2. Viability of embryos.
3. Embryo destruction and relief of human pain.
4. Consent of donors.
127
What are the guiding principles for hPSC research by CHIR?
- 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
What are the types of hPSC research that conformed with the guidelines?
- 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
What are the types of hPSC research that would not conform with CIHR guidelines?
- create human embryo to derive stem cells
| - research involving cloning
130
Describe how stem cell therapy is done
* 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
What are the challenges/requirements for mass production of hPSCs?
- 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
How is GMP cell banking done for an iPSC product?
- 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
What are the reasons 2D cultivation of stem cells is not popular?
- 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
What is the advantage of the 2D multi-plate single-use culture system with pH, DO, and temp control?
this approach mimics the 2D plates in which the cells are first cultured but allows scale-up
135
Why is 3D (stirred tank bioreactor) cultivation of stem cells the best approach?
- 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
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?
- 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
What is the most used culture method for stem cells?
repeated batch
138
What are the issues of perfusion of stem cells?
while it has the highest cell yields and highest contamination risk it has limited applications
139
What are the biggest challenges with regards to commercial stem cell processes?
- Aggregate size control.
- Potential for cell differentiation.
- Monitoring pluripotency during a run
140
What is some stem cell therapy nearing therapeutic use?
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
What are organdie cultures?
- 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
What is multiple sclerosis?
autoimmune disease of the central nervous system
143
What are the current multiple sclerosis treatments?
- Interferon beta (suppresses immune attacks)
| - High doses of adrenocortical steroids
144
What happened the stem cell therapy trials using mice with MS?
- 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
How does phage therapy differ from broad-spectrum antibiotics?
don’t wipe out beneficial bacterial flora in the intestinal tract
146
Why was there a resurgence in phage therapy?
due to the rise of antibiotic-resistant bacteria
147
What are the 2 of E. coli (coliphage) phage from the family mycoviridae?
- T4
| - lambda
148
What is of E. coli (coliphage) phage from the family inoviridae?
filamentous phage
149
What are lytic phage best for?
anti-bavterial therapy (reduce lateral genetic transfer)
150
What is the issue with phage that impacts purification?
phage are very small in size
151
What can the phage genome be like?
- DNA, RNA
- single-stranded, double stranded
- circular, linear
152
In the mechanism of action of lytic bacteriophage, what causes lysis?
- amurins – inhibit synthesis of peptidoglycan to destroy cell wall.
- two-protein system (Holins & Endolysins
153
How did Hershey & chase confirmed the means by which T2-phage infect bacteria?
- 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
How is bacteriophage produced in culture?
- 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
What is used to capture and purify phage>
DEAE (AEX) Disk monolith column Chromatography
156
Why is DEAE (AEX) Disk monolith column Chromatography used to capture and purify phage?
- 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
What are the polishing steps of phage capture and purification?
remove residual DNA & HCP, endotoxins etc.
158
What in the human immune system can inhibit phage effectiveness?
Anti-Phage Neutralizing antibodies
159
What are the ways to deal with reduced efficacy of phage therapeutics owing to anti-phage antibodies?
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
How does the phage and immune system work in synergy?
- 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
What is the phage resistance in bacteria like?
bacteria can evolve to resist phage but phage evolve at a faster rate than biochemists can modify or create new antibiotics
162
What are the challenges associated with phage therapy and solutions?
- 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
What are some novel variations on phage therapy in development?
- prophylactic phage therapy - oral admin
- reduce pathogen levels in poultry
- general disinfectant
164
What are therapies in development with regards to phage?
personalized phage therapy for patients with very tough infection
165
What are some obstacles of commercializing phage therapy?
- cannot patent natural phage
| - no established regulatory pathway for drug approval
166
What is the present day status of phage therapy?
- not approves for use in humans yet
| - approved for use in food safety and agriculture
167
What are T cells?
- 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
What are T cells limitations to cancer?
- 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
What are chimeric antigen receptor (CAR) T cells?
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
What is CAR T-cell immunotherapy?
- 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
Compare the structure of natural TCR vs. CAR in T-cells
- 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
What is the difference of how TCR and CAR bind cancer cells?
CAR T cells do not require costimulation that normal T cells require and know cancer cells can hide their Ag
173
Compare CAR- and TCR-engineered T-cell characteristics
- 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
What is avidity?
binding strength measurement
175
What is cytokine release syndrome (CRS) associated with CAR T-cell therapy?
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
What is the rationale of creating universal CAR T-cell therapies?
- Using allogeneic donors or “universal” T-cells.
- Simplified manufacturing.
- Increased drug availability + lower cost.
- “Off-the-shelf” CAR T-cell therapies
177
What is the rationale of creating universal CAR T-cell therapies?
- Using allogeneic donors or “universal” T-cells.
- Simplified manufacturing.
- Increased drug availability + lower cost.
- “Off-the-shelf” CAR T-cell therapies
178
What is the potential of creating a universal CAR T-cell therapy?
- “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
What are some examples of diseases tested using CAR T-cell therapy?
- solid tunours
| - multiple myeloma (CD19)
180
What is the benefit of using CAR T-cell therapy along with chemo?
Chemo attacks B cells and reduces competition that improves the ability of the CAR T-cells to expand and multiply
181
What are the challenges of CAR T-cell product commercialization?
- Understanding long-term safety.
- PRICE: the very high price raises questions.
- Anticipating global regulatory concerns & unfolding changes with time
182
What are the opportunities of CAR T-cell product commercialization?
-Expanding the type of cancers treatable to include solid tumors
