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BioPharmaceutical Engineering > BPE > Flashcards

BPE Flashcards

1
Q

What are the 4 examples of cell host?

A
  1. Bacteria
  2. Yeast
  3. Mammalian cells
  4. Insect
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2
Q

What is biopharmaceutical engineering?

A

The application of engineering principles to enhance the performance and production of biopharmaceutical products.

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3
Q

What is a signal peptide

A

A short peptide(16-30 amino acids long) present at the N-terminus

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4
Q

What is the N-terminus

A

The start of a protein or peptide

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5
Q

What is the first step in the process?

A

Objective

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6
Q

What does HEK cell stand for?

A

Human embryonic kidney cell.

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7
Q

What is the first step of the design process?

A

Objective

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8
Q

What is the second step of the design process?

A

Design Criteria

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9
Q

What is the third step of the design process

A

Measure

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10
Q

What is the fourth step of the design process?

A

Test

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11
Q

What is the fifth step of the design process?

A

Model

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12
Q

What is the sixth step of the design process

A

Design

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13
Q

What is the seventh step in the design process?

A

Validate

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14
Q

What is the eighth step in the design process?

A

Benefits

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15
Q

What are the 3 structures in the central dogma of molecular biology?

A

DNA, RNA, protein

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16
Q

What process converts DNA to RNA?

A

Transcription

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17
Q

What process converts RNA to DNA?

A

Reverse transcription

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18
Q

What process converts RNA to protein?

A

Translation

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19
Q

What are the products when cells are used as factories?

A

RNA, Proteins

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20
Q

What are the products when the cell is used as the product?

A

Cell therapies

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21
Q

What are the 3 parts of DNA?

A

Promoter - Coding sequence Ancillary DNA

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22
Q

What do promoters do?

A

Dictate the kinetics of gene expression

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23
Q

What are motifs?

A

Building blocks of DNA that bind proteins, interact with receptors and form secondary structures

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24
Q

How can DNA be redesigned?

A

Combining motifs in optimal order

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25
Q

What are the objectives for DNA in manufacturing?

A

Optimising expression levels, minimising silencing and instability, and minimising off target effects.

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26
Q

What are the objectives when using DNA as products?

A

Increase specificity/ reduce off-target, optimise gene expression, Minimise immunogenicity, Max half-life.

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27
Q

What is the structure of mRNA?

A

5’UTR - Coding Sequence - 3’UTR (Poly A Tail)

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28
Q

Which parts of the mRNA control the level of expression?

A

ALL 3!

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29
Q

What controls the ‘other functionalities in DNA and mRNA?

A

Generic DNA the coding sequence.

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30
Q

How is mRNA redesigned?

A

Using multi-parametric optimisation, might do 500 base pairs in one go

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31
Q

What is a sliding window?

A

A tool used in mRNA engineering that can help optimise 50 base pairs at a time, as opposed to working with the whole sequence

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32
Q

What is mRNA engineering all about?

A

Avoiding making the mRNA ‘bad’

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33
Q

What are the goals when using mRNA for manufacturing?

A

Maximising expression levels and minimising off target effects on the cell factory

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34
Q

What are the goals when using mRNA as products?

A

Maximising gene expression level in target cell, Minimise immunogenicity and maximise half-life.

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35
Q

What is the structure of a protein?

A

Signal Peptide —> Protein sequence

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36
Q

What do signal peptides control?

A

Protein transport/expression rate

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37
Q

What controls the functional effect of a protein?

A

Protein sequence

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38
Q

What is a protein?

A

A string of amino acids

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39
Q

What dictates how a protein works?

A

The secondary structures produced from amino acid folding.

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40
Q

What are the building blocks in protein engineering?

A

Motifs that interact with proteins, secondary structures that interact with proteins/receptors/DNA/mRNA & perform functions.

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41
Q

What part of the protein is used when it is being used for manufacturing?

A

Signal peptide

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42
Q

What are the goals when using proteins for manufacturing?

A

Optimising expression levels, minimising off target effects.

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43
Q

What are the goals when using signal peptides as products?

A

Maximise gene expression level in cell.

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44
Q

What are the goals when using proteins as products?

A

Maximise activity / effectiveness,, minimise off target effects, encode cell type specificity

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45
Q

What are the type of products relevant to this course?

A

DNA, mRNA, proteins, cells

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46
Q

What is the goal in the objective step?

A

What are you trying to achieve, value statement. All tech should be driven to achieve a measurable target.

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47
Q

What is the goal in the design criteria step?

A

What features will the engineered biological component/technology need to have?

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48
Q

What are the 2 types of measuring technique?

A

Top-down, bottom-up

49
Q

Explain top-down measuring, simply

A

Want to understand the whole system

50
Q

Explain bottom-up measuring, simply

A

Want to understand individual building blocks

51
Q

What question are you answering in the measure step?

A

What do you need to quantify?

52
Q

What are OMICS?

A

Methods of measurements including genomics, proteomics, metabolomics, metagenomics and transcriptomics.

53
Q

What is transriptomics?

A

A measurement techniques that analyses which genes are expressed and to what level

54
Q

What is proteomics?

A

Measurement technique that analyses what proteins are in the cell

55
Q

What questions are you answering in the test phase?

A

How do you test hypotheses about how the system works?, How to obtain data that expands knowledge of system working

56
Q

What is the aim of the model step?

A

Building a model that facilitates design of new biological components.

57
Q

What are you answering when modelling

A

How much data do you need? Can you minimise the number of test components?

58
Q

What is the question in the design step?

A

How can you convert a model into designed components? How big does the library need to be to provide a high chance of success while maintaining realistic timings and costs?

59
Q

Explain the Validate step

A

Are you using industry relevant testing?
How many components to take forward?
How to move up TRL scale

60
Q

explain benefits

A

How are you going to describe/sell the benefits of your tech

61
Q

What are the main 3 benefits of biopharma engineering?

A

Increasing speed to market
Reduced cost per dose
Better patient outcomes

62
Q

How is mRNA product made?

A

in vitro transcription, using RNase T7 as a catalyst

63
Q

What is the issue with mRNA input components?

A

Expensive, unreliable supply chains

64
Q

mRNA is a simple system, what is the problem with this?

A

May not be able to produce complex products at high quality.

65
Q

What are some things to do when designing mRNA production platform?

A

Improved Quality
High Transcription
minimise cell specific productivity
High cell productivity
High cell growth
Cheap/reliable input materials.

66
Q

What host cell will be used for mRNA manufactue and why?

A

Bacteria, because mRNA is easy to make and bacteria is a simple host.

67
Q

What should be a key consideration when designing mRNA

A

It has a short half life in E. Coli, so key design criteria could be enhancing mRNA stability in E. Coli

68
Q

What can we engineer when a cell is producing mRNA?

A

How much mRNA the cell makes, how quickly it degrades.

69
Q

What could increase stability in mRNA?

A

A more complex structure such as a circle,as then the enzyme can’t digest/degrade it.

70
Q

What makes a good control system?

A
  • Representative of industry
  • Not purposefully ‘hobbled’
  • Well understood and recognised
71
Q

What are the 2 types of test?

A

DoE and OFAT

72
Q

what does DoE stand for?

A

design of experiments

73
Q

what does OFAT stand for

A

One factor at a time

74
Q

What does TRL stand for?

A

Technology Readiness Level

75
Q

What TRL step do most projects fail on?

A

4/5

76
Q

What is TRL 1

A

Basic principles observed and reported

77
Q

What is TRL 4

A

Lab testing of component or process

78
Q

What is TRL 5

A

Lab testing of integrate system

79
Q

What is TRL 6

A

Prototype system verified

80
Q

What is TRL 8

A

System complete and qualified

81
Q

What is TRL 9

A

System proven in operational environment.

82
Q

What can be done to validate past TRL5

A

Scale-up, compare to existing biology Optimisation of processing, Quality analytics

83
Q

What are some validation steps?

A

Manufacture product, Use the product in reactions, Compare to incumbent tech

84
Q

What is a DNA plasmid?

A

A small circular, double stranded DNA molecule, distinct from the cells chromosomal DNA

85
Q

What are some benefits of using DNA plasmids?

A

They are cheap and simple to manufacture, compared to AAV) and relatively stable compared to mRNA, there is also no restrictions on cargo size

86
Q

What are the drawbacks of using a DNA plasmid?

A

It is inherently immunogenic

87
Q

What are dendritic cells

A

A type of cell found in tissues that boosts immune response by showing antigens on its surface to other cells in the immune system

88
Q

What is the function of TNF alpha?

A

Ut is responsible for inflammation and a range of signalling events

89
Q

What are some issues in DNA plasmid design?

A

Immunostimulation causing off target effects Receptors that recognise foreign DNA is poorly understood. DNA plasmids are lost quickly from cells, reducing expression per dose.

90
Q

How can you pick biomarkers?

A

Use bottom up and top down measurements to study markers and see what elicits a response out of the target cell.

91
Q

What is important to show for a new biotech

A

Genes identified are biomarkers
Tool doesn’t produce false negs/positives
Tool accurately identifies small differences immunostimulatory behaviour.

92
Q

What could you do to validate a technology?

A

Get companies to test designs in the new tech and then in patients.

93
Q

What are some design criteria when engineering DNA gene therapies?

A

Reduce activation of immune response
Whilst keeping gene expression and manufacturability the same

94
Q

What is a homotypic library

A

A data set created by trying the design criteria one at a time

95
Q

What is important about heterotypic libraries?

A

They need to be systematic to test design hypotheses

96
Q

How big should the library be?

A

Large enough to decipher required rules but small enough to fit cost & time objectives.

97
Q

What happens if a DNA gene therapy exists in a patient’s genomic DNA?

A

Cancer

98
Q

What are some design criteria for a DNA gene therapy

A

Boosted early expression/retention
Increased plasmid retention rate
Boosted Late expression

99
Q

What does recombinant mean?

A

relating to or denoting an organism, cell, or genetic material formed by recombination

100
Q

What cell is used for most recombinant protein production?

A

CHO cell factories

101
Q

How are proteins made using CHO cells?

A

DNA is inserted into the cell genome that encodes for the expression of a new product. Followed by selection of a ‘top’ clone which is used in large-scale bioreactor to produce kg quantities.

102
Q

What are limitations of CHO cells

A

Most CHO cell alternatives are good at making ‘vanilla’ products but struggle with next-gen products.

103
Q

What are the key issues with CHO cell factories?

A

Low productivity, cell death, poor product quality and aggregation

104
Q

What are the limitations of in vitro protein production

A

Hard to scale, cant handle next-gen, high costs

105
Q

Benefits of in vitro protein production?

A

It would be more standardised and predictable. also more engineerable.

106
Q

What is qP

A

Cell specific recombinant protein production rate

107
Q

Design criteria for CHO cell line

A

Increase qP, maintain high cell growth rate, Improve product quality.
Extras: INcreased average clone perfomance

108
Q

What is measured in CHO cells?

A

Genes, introducing and deleting

109
Q

What OMICs are done in CHO cell line development

A

Almost always transcriptomics

110
Q

What is a phenotype

A

The characteristics that the cell exhibits

111
Q

What is a signal peptide?

A

A short amino acid sequence that controls the rate of translocation(movement of a protein through a cell)

112
Q

What percentage of protein’s have a signal peptide?

A

about 35% - All vaccines, cell therapies and recombinant proteins

113
Q

What is the DNA expression construct?

A

Promoter - 5’UTR - signal peptide - coding sequence - 3’UTR

114
Q

What is the mRNA expression construct?

A

5’UTR - signal peptide - coding sequence - 3’ UTR

115
Q

Why are people reluctant to manufacture signal peptides?

A

As opposed to DNA, by messing up you can change the product you are making.

116
Q

What are the 5 problems with signal peptides?

A
  1. Performance is product specific
  2. No-one knows how signal peptides work
  3. Signal peptides are hard to test
  4. Because they are part of the product there is problems with regulations
  5. It is hard to future proof your solutions.
117
Q

What are the historical challenges with signal peptide manufacture?

A

Highly product-specific
Poorly understood
High ‘failure-rate’
Off -target effects

118
Q

How are DNA gene therapies switched on?

A

By entering the cell.

BioPharmaceutical Engineering (1 decks)

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