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Complex Pt 2 > Future > Flashcards

Future Flashcards

1
Q

The quality triology

A

Quality planning
- to plan and define the quality req.m of end product
Q control
- the variability in product and process attributes need to be controlled by scientific/ risk-based statistical tool for monitoring end product quality
Q improvement
- conti. process throughout product life cycle by regulating controls

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2
Q

5 steps of Quality by Design

A
  1. Define the quality target product profile (QTPP)
  2. Identify critical quality attributes CQA
  3. Identify critical material attributes CMAs and critical processing parameter CPPs
  4. Design space development
  5. Quality control strategy& continuous improvement
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3
Q

6 control strategy for quality improvement in QbD approach

A

1 Input materials testing
2 Release testing (specifications) on end product or real time release testing
3 Product characterisation
4 In process controls (inc in process materials testing)
5 Continuous process verification
6 Process characterisation and quality risk management/ assessment and justification of CQAs control and of quality risk control

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4
Q

the basic concept of tissue engineering

A
  1. cell isolation and purification of stem cells
  2. cell expansion in numbers (enough to make organ)
  3. seed on a suitable scaffold (3d structure), use gf to stimulate proliferation and differentiation
  4. maturation of tissue in Petri dishes in lab
  5. implantation in patient
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5
Q

4 methods of cell isolation

A
  1. differential adhesion (certain cells bind to certain surface better than others)
  2. density centrifugation (isolate mono nucleus cells)
  3. FACS (fluorescence- activated cell sorting)
  4. MACS (magnetic
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6
Q

3 methods of cell expansion

A
  1. stirred tank (3d envrionement)
  2. fluidised bed (pass culture medium through tube into the cells, fluidise the particles, skyscraper scale!)
  3. hollow fibre (bundle of 3d fibres for cell to grow on, packed in a small vol)
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7
Q

7 characteristics of ideal 3d support (scaffold)

A
  1. biocompatible
  2. biodegradable
  3. cytocompatible (cell need to be able to stick on them)
  4. porous
  5. mechanically appropriate
  6. architecturally appropriate
  7. growth promoting - controlled release of gf
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8
Q

natural scaffold materials

A
  1. polypeptide eg collagen, gelatin, fibrin, laminin (ECM)

2. polysaccharides eg hyaluronic acid, alginate, chitosan

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9
Q

synthetic polymer scaffold materials

A

polyesters are the most commonly used
eg PLGA - poly lactic co glycolic acid
made up of lactic acid and glycolic acid. can control degradation rate of scaffold. if more LA conc, then more hydrophobic, slower degradation vice versa
- bioceramics and bioactive glasses eg hydroxyapatite. for osteoblast growth into bones
- decellularised tissue (use donor tissue and remove cellular components so no rejection then grow pt cell on them)

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10
Q

the advantages of having synthetic rather than natural polymers for scaffold

A

controlled parameters eg degradation rate, strength, chemical and mechanical functions, adhesion rate

  • reproducibility - scalibility
  • bulk processing
  • interesting properties eg temp responsive
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11
Q

7 method of scaffold formation

A
  1. compression
  2. solvent casting (liquid pour into mould)
  3. particle leaching
  4. freeze drying
  5. spinning
  6. electrospinning
  7. 3d printing
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12
Q

scaffold provides 3d environment for cells to grow - much better results than one plane growth. what are the two 3d scaffold that’s commonly used

A

hydrogel

nanofiber - cells within nanofiber interact with signals by neighbouring cells as well as by the fiber

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13
Q

which arrangement of scaffold fibre can produce longer length of myotube?

A

aligned not random

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14
Q

how does the geometric cue e.g. stiffness, patterning of scaffold affect (stem cells for) bone cell growth

A

the more drilled holes and more random (disorder) the arrangement of fibre surface, the more likely for osteopontin and osteocalcin to grow into bone tissue. muscle tissue is the opposite

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15
Q

one of probs with synthetic material of scaffold is that they are not particularly cell adhesive or growth promoting. 2 methods to resolve this are

A

1/ blend solution - blend sol of polymer with agents that is adhesive to cells (or blend directly w cells eg laminin), eg collagen. then form scaffold
2. chemical plasma and wet chemical treatment, give scaffold functional groups that cells like to attach to. covalently attach to proteins, eg.make amide bonds through carbo acid and amine fucntional groups

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16
Q

3 key ECM adhesive peptides sequences used to improve the adhesivity of scaffold eg PEG hydrogel scaffold

A

1 add RGD (arg-gly-asp) to promote ingtegrin receptor interactions with cell surface improve adhesion
2 YIGSR Tyr-Ile-Gly-Ser-Arg- lamini ECM
3 IKVAV Ile-Lys-Val-Ala-Val- lamini, nerve

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17
Q

we can modify scaffold to improve its ability of attaching to laminin so better formation of nerve tissue. what are the 3 ways? which gives the best nerve tissue growth results?

A

1/ colavent coupling of laminin by adding COOH groups onto nanofiber material
2/ physical adsorption of laminin onto nanofiber mat
3/ electrospun blended laminin polymer nanofiber mat
blended

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18
Q

compare static vs perfused bioreactor in bone defect repair

A

Bioreactor culture increased cell proliferation and bone matrix formation in comparison to static
-Patient-specific bone constructs a possibility using this technique (ct scan of pt bone structure, design scaffold then use pt own msc to grow)

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19
Q

is body a bioreactor? how does this method work for bone growth?

A

yes. body has the correct environment for tissue development eg chemical signalling, sheer forces.
- you inject alginate hydrogel into space bw periosteum and bone to activate stem cells cause proliferation and migration of cells into the gel matrix to form new bones
- use cell-free scarffold to stimulate repair

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20
Q

is acellular strategies better than engineered cellular repair?

A

yes - can be designed to release cocktails of small and macromolecular drugs and to recruit specific cells (avoid isolation and enrichment etc)
optimum envrionemtn for tissue growth

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21
Q

the challenge of rebuilding capillary bed is the complexity in the vascularisation and the fine size of the each vessel. what are the 3 solutions

A
  1. seed scaffold with endothelial cells RANDOMLY.
  2. incorporate VEGF into scaffold, implant into pt and attract pt own endo cells hope to make the right cap
  3. build scaffold from donor around vascular bed ex vivo (in-vitro)
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22
Q

which component of blood contains important proteins eg GF for blood vessel formation?

A

platelets contains VEGF. PDGF

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23
Q

what the endothelial progenitor cells in our blood that can form blood vessels when stimulated?

A

endothelial colony forming cells ECFCs

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24
Q

what are the steps to produce a capillary vasculature

A
  1. centrifugation of blood to obtain plasma
  2. plasma rich in GF that can form capillary bed
  3. signification of plasma to form human platelet lysate
  4. HPL is a sol full of GF and fibrin
  5. add endothelial progenitor cells -endothelial colony forming cells ECFC into lysate
  6. maturation
  7. formation of bv
  8. GF stimulate pt’s existing vessel to integrate w new bv
  9. pt’s own endo cells, so no rejection
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25
Q

newt can regenerate lost limb, how can mammal regenerate tissue with the help of pharmacological treatments?

A

myoseverin can de-differentiate human muscle into mononuclear myoblast cells

  • add reversine to proliferate and send cells to a even earlier more plastic state where they have more potential to diff (can diff into fat, muscle, bone - multipotent)
  • cells fuse together then re-differentiate
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26
Q

what does quiescent mean for stem cells?

A

losing the ability to be activated, proliferate, differentiate and repair

27
Q

what is quiescent state (of stem cell) in term of signalling molecules present and how can we re-activate those cells

A

quiescent is an inflammatory state, where you can find inflammatory markers signalling in niche, preventing neural stem cells to activated and repair.
- if inhibit the inflammatory markers in stem cell niche of old animal, then we can make them act as if they were young stem cell.

28
Q

how to bioprint a heart?

A
  1. MRI for human heart histology and blood sample collected from pt
  2. blood cell converted to iHPSC
  3. cell differentiation into cardiomyocytes
  4. bio-ink hydrogel. cells combined w nutrients in a liquid hydrogel envrionemtn to keep them alive
  5. 3d bioprinter cartridges are those hydrogel w viable cells
  6. printing one layer at a time guided by MRI 3D shape
  7. put heart into bioreactor that mimic the nutrient and o2 conditions inside human body
  8. cells w fuse into network form living tissue, HEART BEAT
  9. scaffold dissolve leave only heart in bioreactor
29
Q

what is the kaizen (6 sigma process)

A

Kaizen is a continuous process which involves repeatedly making small improvements to the product and production process to ensure that the quality standards are always improving.

30
Q

name the 5 types of bioreactors

A 
stirred tank 
rotary cell culture NASA
compression 
tension
perfusion
31
Q

challenges of tissue engineering

A
  • Stem cell differentiation
  • Vascularisation (leaf scaffold?)
  • Anastomosis (connect between tubes, fibers)
  • Innervation (to supply with nerves)
  • Synaptogenesis
  • Arrangement of multiple cell types in complex 3D structure (bioprinting, layer by layer)
  • Regeneration
32
Q

challenges of 3D bioprinting

A
  1. cell density
  2. resolution
  3. vascularisation
  4. innervation
  5. mechanical integrity
33
Q

what does it mean by planning quality target product profile (QTPP)

A

plan the quality characteristics of target FINAL product before development even begins.
this primarily involve vital final product features that need to be monitored throughout product development
features inc IN VIVO targeted performance/behaviour eg dissolution, dose, appearance, content uniformity, hardness.
what is important to PATIENT

34
Q

what are step 2: Critical Quality Attributes (CQAs), and how they link to QTPP

A

CQA are indicators of product quality performance which directly relate to QTPP. for each process step, an intermediate CQA need to be identified, this may impact the CQA of the final product (drug) = QTPP

35
Q

what does Step 3: Identification of critical material attributes (CMAs) and processing parameters (CPPs) help you achieve in manufacturing drug?
how are they linked to CQA?

A

understanding of the material (API, excipient, device) and the process steps, which in turn aid product understanding.

  • ensure consistency and control of product quality
  • a continuous, marginal gain approach
  • combination of CMA and CPP can determine and control the CQA in the final product
36
Q

the purpose of Step 4: Design Space Development

A
  • allow to explore the relationship bw material, process inputs and CQAs
  • allow manufacturers to show regulatory bodies ‘a comprehensive understanding of their processes’
  • reg bodies approve a design space within an acceptable operational range (for CQAs)
  • allow companies flexibility to operate within the range w/o frequent submission of revised info to reg bodies.
37
Q

describe the flow of manufacturing process from CPP and CMA

A
  1. CPPs and CMAs to QRM (quality risk mgn)
  2. QRM to DoE (design of experiments)
  3. DoE to QbD
  4. QbD back to CPPs and CMAs
38
Q

in QbD, what does company’s quality control strategy allow them to achieve?

A

quality control strategy enable continuous improvement

39
Q

what are control strategies (QbD)

A

a planned set of controls, derived from currently product and process understanding that assures process performance and product quality

40
Q

two example of the controls you can have in control strategy

A
  • in process measurement

- end product testing

41
Q

7 Benefits of introducing QbD

DCCT FML

A

1 reduce develop.m time
2 minimised product cost by multiple times (not individual designed process)
3 helps in effective troubleshooting
4 facilitates timely launch of product
5 increase consumer generic acceptance
6 monitors regulatory oversight (failure) with greater flexibility
7 maintains product life cycle with greater ease

42
Q

What is Quality by Design (QbD)?

A
  • align w kaizen principles
  • aim to deliver products w target quality by planning not by chance
  • building quality into the product by design, avoid quality by testing (traditional)
  • enable conti. improvement and achieve operational excellence
43
Q

describe a typical batch manufacturing process, how long does process take

A

in region 1: synthesis and crystallisation
ship to region 2: blending, granulation and sizing, tablet press& coating
takes months

44
Q

Advantages of Continuous Manufacturing (CM)?

A

1 integrated process with fewer steps (no shipping):
- shorter processing times (all steps undertake at same place)
- increased efficiency
- no manual handling, increase safety
2 smaller equipment and facilities
- more flexible operation
- reduced inventory level
- lower capital costs, less work-in-progress materials. smaller ecological footprint
3 on-line monitoring and controls strategies needed for increased product quality assurance in real-time
- real time release testing provide consistent quality
4 significant reduction in COST
5 the science exists to enable CM of pharmaceuticals
6 no regulatory hurdles for implementing CM (no need to define batch size)
7 CM consistent with FDAs QbD effort - more modern Manu approach - potential to improve QA and drug consistency - enable quality to be directly built into process design

45
Q

describe what a hybrid manufacturing process

A

it is a system consists of two separate ‘multiple disconnected batch steps’:
1 from chemical to API (manufactured in Free Trade Zones around the globe)
2 from API to tablets

46
Q

what are FTZ and what benefit does it provide to Pharma companies

A

free trade zones around the globe. allow companies to import and export goods w/o customs , excise duties/ good and service tax (GST)

47
Q

what are the first two drugs switched from batch to CM process

A

vertex’s orkambitm (ivacaftor)- for CF

Prezista (darunavir) for HIV

48
Q

current challenges for hybrid manufacturing

A
  • need for integration of analytical tool (NIR Near Infrared for blend uniformity) to control system, support feedback, feed-forward control
  • need data mgt tools
  • define representative sampling to consistently assure product quality
    eg location of sampling probes and sample size/frequency
  • need for enhanced process understanding eg
49
Q

state of control requirement for CM is dependent on…?

A

state of control depends on the control strategy implementation

50
Q

3 levels of state of control for CM

A

level 3 - low detectability for addressing natural variance in CM w/o sig end product testing
level 2 - operation within estb. ranges (multivariate) and confirmed w final testing (flexible CMA&CPP within design space)
level 1 - active control sys w real time monitoring of process variables/CQAs, flexible CPPs to respond variability in CMAs

51
Q

what’s the key aims of control strategies in CM

A
  • to monitor in process variability

- to detect disturbances in real time

52
Q

the advantages of Portable continuous, miniature and modular (PCMM) manufacturing

A

1 small footprint
2 highly flexible
3 use an integrated process control system
4 designed for multiple products allow rapid changeover
flexible batch size
production-on-demand

53
Q

what are stem cells

A

unspecialised cells that can
self renew
produce more stem cells
differentiate into other cell types

54
Q

process of inducing a somatic cell into iPSC

A

transfection of fibroblast w/ combination of 24 genes known to be important in ESCs. SOX2 OCT4 NANOG

55
Q

stem cells have a number of transcription factors and cell surface markers, what methods are used to determine this expression?

A

western blotting
RT-PCR
immunocytochemistry (ab)
flow cytometry

56
Q

3 types of PSC (embryonic)

A
  • endoderm (lung)
  • mesoderm (heart, RBC)
  • ectoderm (skin, neuron)
57
Q

how many days does it take for ESC to mature in the embryo?

A

day 1 zygote

day 5 blastocysts

58
Q

what is directed differentiation , eg?

A
  • use the mechanical properties of cell growth surface to direct stem cell differentiation
  • eg stem cell on hydrogel surfaces w/ different elasticities
  • 10-25kpa surface support self-renewal
  • neurone cells prefer soft gel
59
Q

what determines whether our immune system see cells as self or non-self

A

human leukocyte antigen complex HLA

60
Q

are HLA inherited

A

yes, as haplotypes (half from mum/dad)

61
Q

are hPSCs hypoimmunogenic due to its undifferentiated state?

A

no, hPSC express HLA class 1 antigen, but not class II

62
Q

how to make iPSC hypoimmunogenic?

A

use CRISPR cas9 knock off HLA class 1+2 (gene at 2 loci), use lentivirus to deliver CD47- a tumour antigen provides shielding effect and avoid phagocytosis

63
Q

PSC has teratoma risk?

A

yes

Complex Pt 2 (4 decks)

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