Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Biotechnology > Mammalian Cell Expression of Recombinant Antibodies > Flashcards

Mammalian Cell Expression of Recombinant Antibodies Flashcards

1
Q

Why do we need mammalian expression systems?

A

Post-translational modifications E.g. antibodies Fc region glycosylation

Protein folding:
Primary - Peptide bonds
Secondary - Beta pleated sheets/Alpha helix
Tertiary (conformational structure - decides biological activity): Di-sulphide bonds (between cysteines), Hydrophobicity
Quaternary (some proteins, not all), Globular - joining of protein units E.g. antibodies, Heavy chains (x2), Light chains (x2)

Location of protein creation, Eukaryotic - separation of different parts of process
Nucleus 
Cytoplasm 
Quality control  - ER
Glycosylation - ER + Golgi body
Di-sulphide bonds - ER
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Major Mammalian Cell Lines

A

Chinese Hamster Ovary (CHO) cells
Human Embryonic Kidney (HEK) cells
Murine - Sp2/0 OR NSO
HeLa

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Characteristics of Chinese Hamster Ovary (CHO) cells

A

Quite durable
Can survive variations in conditions
No batch-to-batch variation (or at least v.low)
Easily adapted to suspension/serum-free conditions
Serum introduces unwanted proteins
E.g. endotoxins
Yields - 2-6g/L
Human pathogens cannot replicate in CHO cells
Glycosylation patterns need to be carefully monitored
Why? Avoid anaphylactic response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are Chinese Hamster Ovary (CHO) cells used for?

A 
Monoclonal antibodies
Cytokines
Enzymes
Hormones 
Clotting factors...etc
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Characteristics of Human Embryonic Kidney (HEK) cells

A

Made using adenovirus (retrovirus???) fragments
Yield - 1g/L (LOWER)
Very close match to other human cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are Human Embryonic Kidney (HEK) cell used for?

A

Used for clotting factors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are murine cell lines used for?

A

Make mAbs (monoclonal antibodies) - humanize everything but key binding part

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Vector Systems (Basic)

A

Origin of replication (ORI)

Selectable markers

Antibiotic resistance

Promoter (e.g. CMV used as is a strong promoter)

Multiple cloning sites - increases expression

Termination site

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Types of vector system (other than basic)

A

Two-Vector System

Bicistronic Vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Bicistronic Vector

A

Two genes in one vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Two-Vector System

A

Two vectors in one cell

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How do you make a bicistronic vector?

A

Internal ribosome entry site (IRES), initiated translation independently of the 5’ cap

start-gene1-IRES-gene2-termination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Purification methods

A

Affinity Chromatography

Size exclusion

Ion Exchange Chromatography

Hydrophobic Interaction Chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Affinity Chromatography

A

E.g. nickel-ion affinity chromatography

Column and tags (e.g. His-tags)
Binding at X pH
Elution at X pH
Wash off non-bound proteins/molecules

A good general first step

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Size Exclusion

A

A further way to filter (no usually the first step)
Filtration through a gel

E.g. 150 kDa, smaller than get excluded

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Ion Exchange Chromatography

A

Cation & anion

Resin with modified charged functional groups
Selecting for cations-> negative
Selecting for anions-> positive

Based on isoelectric point
pH give protein a neutral charge at X point
Use that pH point to elute

Flow through
Bind to resin and flow through attached

Bind and elute

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are polyclonal antibodies?

A

From hyperimmunization animals

Uses adjuvant - elevate immune response

Made by different B lymphocytes

Bind to different parts of the antigen - multiple paratopes target multiple epitopes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are monoclonal antibodies?

A

Secreted by single B lymphocyte clone - select for cell that produces antibody of interest

One antigen binding site (paratope), recognises a single epitope (antigen binding site on antigen)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What produces monoclonal antibodies?

A

Produced by hybridoma (cells) or recombinant antibody technology (vectors)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Why use polyclonal antibodies?

A

Fast

Inexpensive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Why not use polyclonal antibodies?

A

Cannot engineer, don’t know gene, just produced by immunized animals

Finite - whatever is produced by the animal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What are hybridoma cells?

A

Injecting a specific antigen into a mouse, collecting an antibody-producing cell from the mouse’s spleen, and fusing the antibody-producing cell with a tumour cell called a myeloma cell.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Antibody fragments

A 
Fv
scFv 
dsFv
scAb
Fab - variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Fv

A

Fragment of antibody Variable regions

Variable heavy and variable light, without constant domain (NOT STABLE)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

scFv

A

Single-Chain Fragment of antibody Variable regions

FV +linker (single-chain (sc) linker = cellulose)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

dsFv

A

FV +disulphide bonds (double chain (ds) linker)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

scAb

A

scFV + human constant light domain (stability and detection)

Secondary antibody binds to constant light domain (draw two elements closer together)
Assay detection improvement

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Fab

A

Variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Epitope types

A 
LINEAR 
CONFORMATIONAL
IMMUNO-DOMINANT
IMMUNOGENIC
NEUTRALISING
30
Q

LINEAR epitope

A

RECOGNISES AMINO ACID SEQUENCE

CAN BIND WHETHER PROTEIN DENATURED OR NOT (BASED ONLY ON PRIMARY STRUCTURE)

31
Q

CONFORMATIONAL epitope

A

RECOGNISES TERTIARY STRUCTURE

ABILITY TO BIND LOST WITH DENATURATION

32
Q

IMMUNOGENIC

A

PRODUCE STRONG IMMUNE RESPONSE

33
Q

NEUTRALISING

A

BINDING STOPS ACTIVITY (NOTHING ELSE REQUIRED)

34
Q

EPITOPE

A

Antigen receptor (where the antibody binds)

35
Q

PARATOPE

A

Antibody component binding site

collection of CDRs - variable regions

36
Q

What can you do to improve antibody affinity?

A 
Change size (make smaller)
Improve stability
Subtype switching 
Species switching
Isotype switching
Reformatting
37
Q

Why change the subtype of an antibody?

A

Think about immune system recruitment

IgG types 1, 2, 3, 4…etc, respond to different types of infection

38
Q

Why would you carry out species switching with an antibody?

A

To reduce immunogenicity

e.g. humanise mouse antibodies

39
Q

Why would you reformat an antibody?

A

Don’t want full IgG molecule
Don’t want/need full molecule
Just need binding site/particular ligand

40
Q

Shortcomings of mouse mAbs in therapy

A

Glycans in Fc part considered “foreign” by human immune system

41
Q

In vitro technologies developed to generate fully human antibodies

A

Phage display

Ribosome display

42
Q

How does phage display work?

A

Display protein on surface of filamentous phage

recombinant protein (usually scFV) + P3 gene for filamentous display

43
Q

What is bio-panning?

A

How to get BACTERIOPHAGE DISPLAYING ANTIBODY (contain gene encoding for that antibody fragment)

44
Q

How does bio-panning work?

A

Antibody library of bacterial cells NOT bacteriophages YET (e.g. E.coli containing the vector (e.g. pHEN))
+infect with helper phage N13KO7

Replicates inside bacterial cell and is released with phage particles displaying the antibody fragment on its surface

Then use ELISA as appropriate to isolate bacteriophages

45
Q

Differences between library types

A

Niave = IgM of B cells - first level of response (because it is not responding to anything)
IgM must be affinity matured to IgG, which adds time (~6 months)

Synthetic - DO I NEED TO KNOW THIS???

Immunised = IgG of B cells - second level of response

46
Q

What is a single-domain antibody?

A

Single domain = only heavy chain (does not req. Light chain)

47
Q

Benefits of a single-domain antibody

A

Allows for rapid tissue penetration
Good expression in bacterial and yeast systems (cheaper & more stable (temp./pH))
Can target additional/novel epitopes (antigens)
E.g. shark (IgNARs) and camel (HcAbs)

48
Q

What is ribosome display?

A

RNA fragments expressed on the surface of ribosome

Limited to single-chain display - usually scFv (same as phage)

No bacterial transformation

Very large libraries

49
Q

How does ribosome display work?

A 
Antibody library (of vectors in bacterial cells)
Select for best from that set
Incorporate into ribosome display vector
PCR + display vector 
-> mutagenesis 
Transcribed to mRNA (in vitro)
\+ribosomal complex 
-> protein expressed on surface of ribosomes

Selection - for affinity

Dissociate complex
- take the mRNA for that binder

Reverse transcribe to cDNA

Put back in to ribosomal display vector

Several cycles of selection and mutagenesis possible

50
Q

How to make a transgenic mouse?

A

XenaMouse - embryonic stem cells altered
Human heavy and light loci for antibody production

-> mice that will produce human antibodies in response to immunization

51
Q

What is a biosimilar?

A

Binds to the same epitope (antigen)

Similar efficacy, safety and immunogenicity

Different structure of antibody

52
Q

What are next generation antibody therapeutics?

A

Antibody drug conjugates
Bispecifics
CAR-T therapy (personalised medicine)

53
Q

Antibody Drug Conjugates

A

Specific monoclonal antibody with highly potent cytotoxic agents attached by biodegradable linker

To deliver cytotoxic agent(s)

Why use an antibody? -> Increases specificity of targeting

54
Q

Bispecifics

A

One antibody, two distinct binding arms
Bind to different antigens or epitopes on the same antigen

E.g. re-target T cells to kill tumour cells
1 - binds to cancer cell
2 - binds to recruit cytotoxic T cell

55
Q

CAR-T therapy

A

PERSONALISED MEDICINE

Chimeric antigen receptor - T (cell) therapy

  • Type of adoptive cell therapy (ACT)
  • Chimeric antigen receptor - antibody
  • Engineer T cells from patients

T cells clear tumour cells/cancer cells

  • Make antibody to help T cells to find cancer cells
  • Insert antibody gene into T cell
  • T cells now express chimeric antibody on the surface - bind to specific targets on tumour cells

Put modified T cells back into patient

56
Q

What is ELISA?

A

Enzyme-Linked Immunosorbent Assay

57
Q

How does ELISA work?

A

Antibody immobilised on solid surface

  • direct antigen on surface binds to antibody (of interest & linked to enzyme) -> enzyme activity
  • indirect antigen on surface binds to antibody (of interest) then another antibody (linked to enzyme binds) -> enzyme activity
58
Q

What is affinity?

A

The strength of the antigen-antibody bonding

59
Q

What is avidity?

A

The overall stability of the antibody-antigen complex

60
Q

What type of bonding is between antigen-antibody?

A

non-covalent

e.g. hydrogen bonds, van der Waals (gen.) /ionic bonds/hydrophobic

61
Q

What does chimeric mean in the context of chimeric antibody?

A

Variable light and variable heavy regions are different in some way (e.g. species switching) to the constant regions.

e.g. ximab

62
Q

What is a humanised mouse antibody?

A

zumab

variable heavy and variable light regions have been altered to be humanised (mouse interspersed with human sections - particular areas of immunogenicity)

63
Q

How might you maintain the phenotype(displayed)-genotype(DNA) connection?

A

Ribosome display
Phage display
Yeast Surface Display

64
Q

Post bio-panning formatting

A

Phage display scFV (usually) and so these are then reformatted (once harvested) into mAb/scAb and Fab as appropriate.

65
Q

Construction of a phage display antibody library

A

Select B cells that produce antibodies - extract all RNA

Use primers to locate variable regions (light chain and heavy change)

66
Q

What is a CDR?

A

Complementarity-determining regions (CDRs)

= part of the variable chains in immunoglobulins (antibodies)

= where antibodies bind to their specific antigen

67
Q

What does ADME properties mean?

A

Absorption, Distribution, Metabolism, and Excretion properties of the drug molecule

68
Q

What are ADCs?

A

antibody drug conjugates

- application for antibody technology

69
Q

What to ADCs do?

A

Mode of delivery of cytotoxic agents

70
Q

How might you get mAbs across the Blood-Brain Barrier?

A

Bi-specific antibody that can “piggy-back” on an existing transportation system into the brain

One arm binding engage with receptor-mediated transcytosis (type of cellular transport)

Second arm binds therapeutic target

Biotechnology (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions