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The Medicine Year 3 Sem 1 > Biologics and Insulin > Flashcards

Biologics and Insulin Flashcards

1
Q

Why are biologics considered to be versatile?

A

They can replace disease tissue as well as modifying disease tissue

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2
Q

What are the advantages of biologics over small molecules?

A
  • Versatile
  • Faster to market
  • More specific binding (reduced toxicity)
  • Less frequent dosing needed
  • Blockbuster drugs (£1 Billion+ sales)
  • Lower failure rates in discovery pipeline
  • Function can be changed easily
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3
Q

Do biologics require frequent or less frequent dosing?

A

Less frequent - they have longer circulation times compared to small molecule drugs

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4
Q

How are immunogenic effects of biologics addressed?

A

Humanisation of proteins

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5
Q

How is biologic bioequivalence risk managed?

A

Supportive data for structural and functional characterisation

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6
Q

What is biologic bioequivalence?

A

Demonstrates a same level of risk at same dose

Having 2 small drug formulations that become bioavailable at the same rate and extent after administration at the same dose

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7
Q

In terms of hydrophobicity, when are most globular proteins stable?

A

When the loops with the hydrophobic side chains are buried in the interior of the protein.

Unfolding leads to aggregation and colloidal instability

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8
Q

Are proteins stable at their isoelectric point?

A

Many are, but they still aggregate

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9
Q

Describe the process of protein aggregation

A

Unfolding or partial unfolding of protein required

Hydrophobic side chains are become exposed

Unfolding protein molecules aggregate to form large MW aggregates

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10
Q

What are the potential causes of chemical degradation leading to protein instability?

A

Oxidation
Deamidation
Hydrolysis

May lead to instability then aggregation

  • Exposure of cysteine residues and formation of di-sulfide bridges
  • Exposure hydrophobic regions
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11
Q

What are the factors inducing physical destabilisation of proteins?

A
  • Extremes of pH
  • Shear Forces (high pressure)
  • Air/water interfaces
  • Adsorption to solid surfaces
  • Freezing, drying and re-hydration
  • Elevated temperatures and pressures
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12
Q

How do amino acids stabilise biologic formulations?

A

Preferential exclusion/hydration
Decrease protein-protein interaction
Increase solubility and reduce viscosity

E.g. Arginine

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13
Q

How do polymers stabilise biologic formulations?

A

Competitive adsorption
Steric exclusion
Preferential exclusion/hydration

E.g. PEG

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14
Q

How do polyols stabilise biologic formulations?

A

Preferential exclusion

Accumulation in hydrophobic regions

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15
Q

How do salts stabilise biologic formulations?

A

Hydration
Hoffmeister series exclusion

Dependent on the size and charge of the salt ions

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16
Q

How do surfactants stabilise biologic formulations?

A

Competitive adsorption at interfaces
Reduce denaturation at air/water interfaces

Don’t directly interact with protein but interact with interfaces

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17
Q

How does acylation with a fatty acid stabilise proteins?

A

Increasing binding affinity to serum albumin

  • Albumin has an Fc region which means that it can be recycled
  • Results in longer acting insulin, glucagon and interferon
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18
Q

How does PEGylation stabilise stabilise proteins?

A

Reduces plasma clearance rate - so less frequent administration.
But some proteins can become less active when PEGylated

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19
Q

What is the purpose of surface engineering?

A

To remove sites on protein surface that are likely to cause aggregation

Not well controlled

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20
Q

What effect do mutations have on proteins?

A

They alter surface structure and polarity

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21
Q

What is preferential interaction?

A

Denaturant

  • Co-solute binds to surface of the protein
  • Leads to protein unfolding
  • Higher interaction between protein and co-solute when unfolded
  • Positive conc. difference between local and bulk
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22
Q

What is preferential exclusion?

A

Protectant

  • Co-solute has a lower interaction with protein (but not hydrophobic)
  • Leads to higher concentration of co-solute in bulk than in the solvation shell of the protein
  • Co-solute attracts water to itself and away from the protein
  • Makes the surface of the protein less solvated, making it shrink into itself
  • Stabilises protein
  • Negative concentration difference between local and bulk
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23
Q

Define exclusion

A

The thermodynamic mechanism to explain stabilisation by excipients

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24
Q

What is the relationship between degree of preferential exclusion and chemical potential proportional?

A

Degree of preferential exclusion and the increase in chemical potential are directly proportional to the surface area of the protein

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25
Q

How does the process of exclusion work?

A

Minimises the thermodynamically unfavourable effect of preferential exclusion by favouring the state with the smallest surface area

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26
Q

In terms of chemical potential, what happens to unfolded/denatured proteins?

A

Energy is needed to unfold the protein (due to large SA) therefore folded proteins have a high chemical potential

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27
Q

Do unfolded proteins have low energy?

A

No

They have high energy, they have the potential to go back to their native stative or aggregate.

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28
Q

What happens to proteins at low temperatures?

A

Low temperatures extend shelf life

  • Molecules move slower and therefore probability of collision and aggregate is reduced
  • However, cold denaturation (freezing) may lead to protein damage
  • As temperature drops, solvent properties change (dielectric constant, acid/base ionisation, diffusion rates, solubility of hydrophobic residues)
  • Cold denaturation is usually reversible
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29
Q

What happens when biologics are frozen?

A

Repeated thawing and freezing causes aggregation by pH and concentration changes, and provision of nucleation points for aggregation on ice-water interfaces

  • When water is frozen, may have nucleation (formation of ice crystals)
  • Protein may adsorb onto water crystals and promote aggregation
  • Slow cooling will result in large crystals
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30
Q

What kind of substances are used for cryoprotection?

A

Sugars, polyhydric alcohols, oligosaccharides, amino acids

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31
Q

What is the process of cryoprotection?

A

Works by preferential exclusion, lowering cold denaturation temperature and stabilising osmotic stresses

  • Surfactants prevent interactions at ice/water interface
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32
Q

How should frozen vials be treated when thawing?

A

They should be gently mixed (do not shake!) to produce an even distribution and avoid mis-dosing

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33
Q

How does freezing affect the concentration of the protein in a vial?

A

Concentration gradients are formed during freezing and remain if thawed without mixing

When freezing a vial, the energy comes from the side
and the water freezes

Higher concentration of protein in the centre than the side.

Concentration is also higher at the bottom.

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34
Q

What is the main advantage of freeze drying?

A

Formulations have greater long term stability that protein solutions

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35
Q

What is the disadvantage of freeze drying?

A
  • Proteins go through reversible conformational changes during transition into the lyophilised state
  • Exposes buried regions and makes them prone to aggregation - increases risk of aggregation
  • This can also happen during reconstitution

Reactions and denauturation can continue when lyophilised.

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36
Q

How can denaturation be avoided in lyophilised formulations?

A
  • Refrigerate lyophilised medicines to reduce aggregation rates
  • Ensure that the vial is properly sealed to avoid water vapour absorption.
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37
Q

Describe recombinant human insulin

A
  • Monomer (small protein)
  • Exists naturally in a hexameric structure
  • Hexamer has globular protein structure
  • 2 axial Zn ions connected to 6 histidine side chains
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38
Q

How is fast acting prandial insulin engineered?

A

Mutation of one or more amino acids in protein sequence to disrupt assembly through:

  • Converted from hexameric to dimeric and monomeric
  • Diffuses faster and improves transport
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39
Q

What is the benefit of fast acting prandial insulin engineering?

A

Makes the insulin faster acting on sub-cut administration, there is rapid absorption at mucosal barriers and a rapid response from infusion pumps

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40
Q

What is early basal intermediate insulin formulated with?

A

Protamine - to create suspensions forming crystals, when speed of dissociation and absorption varies in the same patient

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41
Q

What does the long acting insulin glargine need?

A

Dissolution of isoelectric precipitates formed after injection which causes variability

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42
Q

Describe long acting insulin detemir

A

Less variable, comes from fatty acid modification
Reversible stabilisation avoids precipitation and dissolution
Binds to albumin, when absorption rate is only slightly affected by blood levels, it circulates for longer

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43
Q

How are mAbs produced?

A

1) Immunisation
Immunise mice with antigen then isolate antibody produced B cells
2) Preparation of myeloma cells
3) Fusion
B cells fused with myeloma cell in PEG to form a hybridoma
4) Clone screening and growth
Clones screened and selected based on antigen specificity and immunoglobulin class
5) Functional characterisation
Characterise using Elyser. Pick clones which have the desired antibody.
6) Scale up clones and wean off selection agents
7) Expand clones using bioreactiors

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44
Q

What is the disadvantage of mouse antibodies?

A

Cause immunogenic reactions
Cleared rapidly
Due to lack human Fc effector functions (do not recycle protein)

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45
Q

What is a chimeric mAb?

A

It has a mouse variable gene

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46
Q

What is a humanised mAb?

A

Has mouse antigen binding loops (CDRs)

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47
Q

How are fully human antibodies produced?

A

Through mice

  • 4 mouse IgG gene is replaced with human transcends in transgenic mouse
  • Mouse is immunised to raise immune response
  • B cells are selected, a hybridoma is produced and bioreactor cell culture produces human antibodies
48
Q

What happens after antibodies are administered?

A

They distribute mainly within the central compartment, penetration inside cells is limited by high molecular weight and hydrophilicity
Low absorption

49
Q

What is the primary route of mAb clearance?

A

Target mediated elimination
Interaction between mAb and target
The resulting immune complexes are then cleared from the body through reticulo-endothelial system (RES).

50
Q

How can the function of biologics be modified?

A

By modifying AA sequence in CDR region of the biologic

Different structures for each indication (excluding similarly structured mAbs)

51
Q

What are the disadvantages of biologics?

A
  • Higher risk of immunogenic effects (immune response)

- Inappropriate molecular target (also applies to small molecules)

52
Q

Name the different types of biologics on the market

A 
Peptides
Protein fragments
Monoclonal Antibodies (mAbs)
Antibody drug conjugates (ADCs)
Viruses
Vaccines
Lipid Nanoparticles
53
Q

What are biosimilars?

A

Biologics produced as generic medicines once they come off patent
Process varies to get same end product

54
Q

What is the current limitations of the manafacture of mAbs?

A

Sequential process with multiple steps - slow

Large scale manufacturing at one site

55
Q

How does PEG allow fusion of B cells?

A

PEG attracts water molecules (osmolarity) which causes the cell membrane to break

56
Q

What mAb features contribute to its pharmacokinetic action?

A
  • Fc region (FcRn)
    Binds to Fc receptor on target cells.
    Important for antibody recycling (prolongs half-life)
  • Fab region (antigen)
    Charged
    Antigen binding affected by charge/PI (isoelectric point)
    Off-target binding
  • Glycan receptor
    Glycan mediated clearance and tissue distribution
57
Q

How are mAbs administered?

A

IV, SC or IM injections

  • IV has a low conc. of antibody
  • SC has a high antibody conc. and allows self injection
  • SC absorption is variable (20-95%) and is facilitated by lymph system
  • Rate of absorption is slow
  • Maximal plasma concentration 1-8 days following SC or IM injection
58
Q

How are mAbs cleared?

A

Not clearly understood
Eliminated by proteolytic catabolism by lysosomes
Results in free amino acids which are recycled

Other mechanisms include:

  • Target mediated clearance
  • Non-specific pinocytosis
  • Fc gamma receptor mediated clearance.
59
Q

How is the PK/PD relationship of mAbs unique?

A

Their PK is markedly influenced by the biology of the target antigen (TMDD)
Differs from small drugs

60
Q

What is the reason that mAbs have a longer half-life than small molecule drugs?

A

Form pH dependent interactions with FcRn which prevents their renal clearance
Antibodies are then recycled
No renal clearance of intact antibody

61
Q

What receptor is responsible for recycling?

A

Neonatal Fc Receptor (FcRn)/Brambell receptor
Mediates recycling of albumin and IgG
Fc region needed on protein

62
Q

Describe the recycling process

A

1) Fc region of IgG binds to FcRn
2) IgG is endocytosed as vesicle into the monocyte/endothelial cell
3) As vesicle enters the cell, its pH decreases to pH 6, forming an endosome (acidic pH)
5) Non-receptor bound IgG sorted to lysosomes for degradation
6) Bound FcRn-IgG sorted into a recycling endosome
6) pH in recycling endosome increases slowly to approach pH of the blood (7.4) as the endosome migrates out of the cell
7) IgG dissociates from FcRn at physiological pH and is released into the blood

63
Q

Why does the vesicle form an endosome during mAb recycling?

A

Low binding affinity at pH 7-7.4.

64
Q

Why is some mAb degraded during recycling?

A
  • Too much IgG compared to FcRn receptors

- Catabolic degradation produces amino acids

65
Q

Why is FcRn binding affinity important?

A

Critical quality attribute for mAbs
Want to have IgG which binds well to FcRn but is also released when required
Modify affinity by changing AA sequence

66
Q

What factors can affect the recycling of mAbs?

A

1) Fv region may also bind to FcRn and alter interactions
Results in shorter half-life as it only dissociates at higher pH

2) Antibody and antigen complexes are also recycled through FcRn pathway
- Antigen binds to Fab region while the Fc region binds to the FcRn
- Results in accumulation of bound antigens in circulation and extension of the half-life of the antigen

Remove antigen by promoting degradation of the antigen whilst the mAb is being recycled

67
Q

What is the half-life of the different IgG types?

A

IgG types 1, 2 and 4: 21 days
IgG type 3: 7 days

Most have a half-life of 14-30 days
IgG1 most common mAb

68
Q

How can the half-life of mAbs be increased?

A

By increasing its molecular weight

Exceptions are Albumin and IgG1, 2 and 4
They have a small MW but long plasma-half life – this is why they’re used

69
Q

How does glycosylation impact the pharmacokinetics of mAbs?

A

Glycosylation important for the half-life of Fc fusion proteins

Glycosylation is not required for an IgG antibody’s long half- life as all IgGs (natural and recombinant) are already glycosylated

70
Q

How does charge impact the pharmacokinetics of mAbs?

A

Cell surface and receptors are often negatively charged

Changing the isoelectric point (pI) of mAbs is powerful way to improve PK
Extent of charge will affect binding affinity

Usual pI = ~8 (+/- 0.5)
Results in half-life of 20 days

Decreasing pI, increases the half-life substantially (lower clearance)

71
Q

How can you change the charge on a mAb?

A

Glycosylation

Mutation

72
Q

What is the effect of increasing the isoelectric point on half-life? Why?

A

Increasing pI results in a constantly charged molecule which may not detect difference in pH during recycling (from pH 7.4 to pH 7.4)
Results in a large decrease in half-life

73
Q

How do mAbs result in an immunogenic response?

A

Formation of anti-drug antibodies (ADAs)
May cause hypersensitivity responses (anaphylaxis)
Result in increased clearance of drug (drop in mAb conc. in the blood)
Immunogenicity of mAb varies across species (chimeric, human etc.)

74
Q

What are the two types of ADAs?

A

Neutralising or non-neutralising

Neutralising = Bind to the epitope (section where the antigen binds to – Fab) on mAb needed for antibody activity

Non-neutralising = Bind to epitope not needed for activity

75
Q

Describe the dose proportionality of mAbs

A

Non-linear PK at low doses

Linear PK at high doses after saturation of target

76
Q

Describe Fc fusion proteins

A
  • Comprise a protein, peptide or receptor exodomain fused to the Fc region of the mAb
  • Binding to Fc region allows it to be recycled, extending the half-life of the protein/peptide/exodomain
77
Q

Describe the Fc region

A

Typically contains the hinge region usually along with the conserved N-glycosylation site in the CH2 domain

78
Q

Describe Antibody drug conjugates (ADCs)

A
  • mAb acts as drug delivery agent
  • Drug forms cleavable linkages with lysine or cysteine residues on mAb
  • Drug is then released following degradation or enzyme reaction in the cell
  • Used for chemotherapy drugs, immunotoxin, radioisotopes or cytokines
79
Q

Describe Multifunctional antibodies

A

Contain two or more variable domains with specific binding affinity to different antigens

Bispecific formats comprise of IgG like and Fab fragment-based constructs
Binds to two targets
Increases binding specificity
Complex to produce and stabilise

80
Q

Describe Antibody fragments

A

Do not have the Fc region

Most prominent region is Fab (fragment antibody binding )

81
Q

Name the types of Novel formats

A

Fusion proteins
Antibody fragments
Multifunctional antibodies
Antidrug conjugates

82
Q

What are the limitations of novel formats compared to mAbs?

A
  • More complex
  • Synthetic therefore they have decrease in conformational stability and solubility
  • Heterogenous product
  • Decreased stability leads to aggregrates and formation of immunogenic response
83
Q

Give an example of an antibody fragment drug

A

Ranibizumab (Lucentis)

Cephalon

84
Q

Give an example of a fusion protein drug

A

Etarnecept (Fc fusion)
Abatacept (Fc fusion)
Dulaglutide (Albumin fusion)

85
Q

Give an example of an ADC drug

A

Trastuzumab entansine

86
Q

Give an example of a multifunctional Ab drug

A

AFM13

Genmab

87
Q

What is the structural difference between long acting and fast acting insulin?

A

Fast acting: Monomer or Dimer

  • Readily absorbed by the body
  • Monomer has better absorption than dimer

Long Acting: Hexamer
- Hexamer not absorbed

88
Q

What is the dilution factor?

A

The behaviour of the protein once it has been injected into the patient

Increasing dilution factor, reduces concentration of Insulin in the body

89
Q

How does changing insulin size affect its MW? What factors affect this?

A

Increasing size, increases MW exponentially

Hexamer size = 5 - 6nm

Changing pH changes the ionisation of the amino acids on insulin thereby changing the size

Can transition from a hexamer to a dimer etc. and vice versa

90
Q

What type of insulin is preferred for mealtime glycaemic control?

A

Fast acting
Sharp peak in insulin than decreases quickly
Least risk of post-prandial hypoglycaemia

91
Q

What influences the half-life of insulin?

A
  • Antibody recycling
  • Immunogenic response (ADAs)

Increasing ADA results in decreasing antibody levels

92
Q

How do ADAs reduce antibody levels?

A

ADA complexes the antibody and two reactions can form:
- Macrophages recognise complex and destroy it
OR
- Complex prevents antibody binding to FcRn receptor and will be degraded by cell

93
Q

Explain how the circulation half-life is related to the dissociation constant (KD) of Fc fusion proteins-FcRn interaction

A

High dissociation constant (KD) means low binding affinity between protein and FcRn
Will be eliminated faster and will not be recycled

94
Q

Explain why chimeric antibodies are not affected by FcRn dissociation

A

Chimeric antibodies have a variable region

Allow Fv antigen binding

95
Q

Explain why chimeric mAbs may be eliminated faster than human and humanised mAbs

A

ADA – immune reaction

96
Q

Explain why Fc fusion proteins have a lower half-life

A
  • Fc fusion proteins have different shape than whole mAb
  • More rigid structure
  • Hinders ability to binding to receptor due to steric hinderance
97
Q

Describe the structure of proteins

A

3D conformation
Dynamic
Relatively flexible
The presence of salt bridges “rigidifies” the conformation

In solution, the hydrophobic AA is buried in the protein core and the hydrophilic AA in the shell

98
Q

Describe the stability of proteins

A
  • mAbs (and other proteins) are not colloids
  • They have a non-uniform distribution of charge
  • DLVO theory used to indicate its behaviour
  • Need to consider attractive and repulsive forces but also hydrophobicity
  • As well as pH, buffer, salt (concentration and type) and concentration of co-solutes
99
Q

What factors affect aggregation?

A 
pH
Temperature (T)
Pressure (p)
Ionic strength
Concentration of co-solutes
  • All can result in unfolding the protein thus ultimately aggregation
  • Reduces reversible aggregation in favour of irreversible aggregation
100
Q

How does pH influence aggregation?

A

Shifts of pH towards isoelectric point or high ionic strength tend to favour aggregation

101
Q

How does temperature influence aggregation?

A

Decreasing temp, decreases rates of aggregation

102
Q

How does pressure influence aggregation?

A

Pressure promotes protein unfolding close to interfaces

  • Most of the proteins will be injected into the patient
  • Pressure is applied during the act of injecting and this can lead to protein unfolding
103
Q

How do co-solutes influence aggregation?

A

Surrounds the protein and draws water away from it, causing it to shrink (fold)
Decrease aggregation

104
Q

How do needles for injection affect the stability of a protein?

A

Coated in silicon

Silicon promotes protein adsorption and aggregation to the needle

105
Q

What is the melting point of proteins?

A

40-50C

106
Q

Proteins should be shaken. True or false?

A

False.

  • Rapid agitation leads to formation of foams (liquid/air interface)
  • Unfolded/Partially unfolded protein may migrate to interface and aggregate
107
Q

Give examples of co-solutes that cause preferential interaction

A

Urea
Guanidine hydrochloride

  • Work at different concentrations
  • Large amount of urea needed to complete unfolding (8mol/L)
  • Much less guanidine hydrochloride needed
108
Q

Give examples of co-solutes that cause preferential exclusion

A

Sucrose

Arginine

109
Q

Name the types of stability testing

A

Stability testing differs from other medicines

  • Accelerated studies (support to establish the shelf life)
  • Long term stability (determines shelf life)
  • Stress studies (Reveal patterns of degradation)
110
Q

Give a summary of the conditions used for stability testing

A
  • Do not go above 40C as protein begins unfolding above 42C (dependent on the protein, some unfold above 50C)
  • High humidity (65-75%)
  • Long term testing for 0.5 – 5 years for most biologicals
111
Q

What types of solutions is protein lyophilisation used for?

A

Low concentration solutions in hospital (1-10mg/ml)

112
Q

What is the process of freeze drying?

A

1) Freeze: product is completely frozen in a vial
2) Vacuum: product is placed under deep vacuum well below triple point of water
3) Dry: heat energy is added causing ice to sublime

Results in dry product of the proteins

113
Q

What is the relationship between protein degradation and turbidity?

A

Increased degradation → unfolding → aggregation → high MW aggregates → turbidity

114
Q

How does sucrose concentration affect protein aggregation?

A

Sucrose is preferentially excluded because it has no hydrophobic groups therefore doesn’t interact with the protein

Prevents protein unfolding and therefore aggregation by attracting water to itself (hydrophilic) which makes the protein shrink into itself

This means more energy is needed to destabilise the protein (high chemical potential) and therefore formation of aggregates is less favoured at high concentrations of sucrose

115
Q

Why is it not good practice to weigh out frozen product/sufficient liquid needed?

A

Protein concentration is not evenly distributed throughout vial

The Medicine Year 3 Sem 1 (7 decks)

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