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Pharmacy Y3S1 > Biologics and Biotherapeutics > Flashcards

Biologics and Biotherapeutics Flashcards

1
Q

What are the differences between biologics and small molecules?

A

Biologics are versitile - they replace and modify diseased tissue
Unspecific binding not applicable with therapeutic proteins (less side effects)
Blood levels and duration of action appropriate to mAbs
Less frequent dosing of mAbs as they have longer circulation times
Do not need different structures of mAbs for each indication
Inappropriate molecular target applies to both
Immunologic effects are a higher risk for therapeutic proteins

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2
Q

What are the production stages of biologics?

A 
Immunization (spleenocytes)
Preparation of myeloma cells 
Fusion (PEG)
Clone screening and picking (antigen specificity and immunoglobulin class)
Functional Characteristics
Scale up and wean
Expansion
Then go on to globalized manufacturing = robots
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3
Q

How were Human Antibodies developed?

A

From a mouse - immunologic reactions and rapid clearance occurred due to lack of Fc receptor functions
Chimeric - mouse variable region (Fv antigen binding via mouse cells)
Humanised - mouse antigen binding loops (CDRs)
Then fully human antibodies produced via mouse

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4
Q

What are the ways in which antibodies work?

A

CDC - Complement Dependent Cytotoxicity
Conjugates - antibody with attached cytokines/toxins causes cytotoxicity attached
Apoptosis induction
Receptor/ligand blockade - cell-cell blocking so nothing can be produced
ADCC - antibody dependent cell-mediated cytotoxicity - attaches to cell then neutrophil binds

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5
Q

How do mAb features contribute to PK?

A

Antigen - target mediated disposition; charge/PI mediated clearance; can lead to off target binding
Glycan receptor - glycan mediated clearance and tissue distribution
FcRn - IgG recycling for long half life

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6
Q

How are biologics eliminated?

A

Not fully understood
Proteolytic catabolism by lysosomal degredation
Target mediated clearance - interaction of mAb and targer then immune complex cleared by reticulo-endothelial system (RES)
Non-specific pynocytosis

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7
Q

Why is the PK/PD relationship unique in biologics?

A

PK of mAbs markedly influenced by biology of target antigen

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8
Q

What is the process of mAbs recycling?

A

Neonatal Fc receptor (FcRn) mediates recycling of albumin and IgG
(Fc region binds to FcRn)
FcRn binds IgG in acidified endosome (pH 6)
Sorting of Fc-IgG complexes occurs
IgG taken up bu monocytes/endothlial cells and endocytic mechanisms and dissociates at physiological pH
OR
Non-receptor bond proteins are degraded in the lysosome to amino acids

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9
Q

What problems can occur during mAb recycling?

A

Fv region may also bind to FcRn so dissociation only at higher pH resulting in a shorter half life
Antibody-antigen complexes also recycles by FcRn pathway can result in accumulation of bound antigens in circulation and longer half life

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10
Q

How long are the half lives of IgG’s 1-4?

A 
1,2,4 = 21 days
3 = 7 days
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11
Q

What is glycosylation important for and not required for?

A

All IgG’s are glycosylated
Important for Fc fusion proteins
Not required for IgG’s long half life

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12
Q

Why do Fc fusion proteins have a shorter half life?

A

Lower binding affinity to FcRn
Glycan mediated disposition
Receptor mediated response

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13
Q

What is the PI of mAbs and why is charge important?

A 
Approx 8 (+/- 0.5)
If constantly charged, mAbs may not feel change to acidic conditions in lysosomes 
Longer half life if PI is higher
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14
Q

How may mAbs cause immunogenicity?

A

Anti-drug antibodies (ADA’s) may form and bind to mAb
Immune complexes form impacting PK, PD, safety and efficacy of mAbs
Hypersensitivity reaction responses: anaphylaxis, infusion reaction, accelerated clearance of drug

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15
Q

Why might conc of mAb drop in blood?

A

Presence of ADA’s and higher clearance

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16
Q

Which factors influence mAb PK?

A

Binding - specific to targer. Binding to FcRn and recycling contributes to half life.
PK/PD - usually dependent on biology of target.
Dose proportionality - non-linear at PK at low doses and linear at higher doses after saturation.
Distribution - partitioning of blood to tissues is 5-15%
Metabolism - catabolism by proteolyutic degradation
Excretion - no renal clearance of intact antibody
Immunogenicity - Formation of ADA against mAb.

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17
Q

Can animals predict human immunogenisity?

A

No. Immunogenicity of mAb varies across species.

18
Q

What are novel antibody formats clustered in?

A 
Antibody fragments
Fusion proteins
Antibody drug conjugates
Bispecific - modified IgG's 
Multispecific antibodies with multiple antigen binding sites
19
Q

Describe antibody fragments

A

Skip Fc part and keep the rest
Fab most prominent `
ScFv is one - single domain antibodies made of VH flexibly linked to VL

20
Q

Describe fusion proteins

A

Comprise of a protein, peptide or receptor exodomain fused to the Fc region of the mAb
The Fc region has the hinge region and conserved N-glycosylation site in the CH2 domain

21
Q

Describe Antibody-Drug Conjugates

A

mAbs used to deliver drugs: chemo, immunotoxins, radioisotopes, cytokines
A cleavable linker in either Cys or Lys residues allows drug release

22
Q

Describe multifunctional antibodies

A

Contain 2 or more variable domains with specific affinity to bind different antigens
Made of IgG like and Fab fragment constructs

23
Q

Where should the amino acids in mAbs/therapeutic proteins be? Which are more highly buried?

A

Hydrophobic in the core = non-polar

Hydrophillic in the shell (outside)

24
Q

How is the 3D conformation formed and why is this important?

A

Salt bridges rigidifies
Unfolding may lead to aggregation - denature/exposure of hydrophobic core. Cannot refold so attached to another to get away from water

25
Q

What factors need to be considered for stabilisation?

A

Attractive and repulsive forces
Hydrophobicity
pH, buffer, salt, co-solutes

26
Q

What is stress and what are examples?

A

Anything that can lead to a conformational change
Increasing T or p
= more irreversible aggregation
= p promotes protein unfolding close to interfaces
Changing pH alters charge
= pH shift towards IP
= increase in ionic strength
= electrostatic forces felt shorter differences

27
Q

How does chemical degredation lead to protein instability?

A

Oxidation, deamidation, hydrolysis
May cause instability then aggregation
Cysteine residues (-SH) exposed or disulfide bridges formed (-S-S-)
Exposure of hydrophobic regions

28
Q

How does physical destabilisation lead to protein instability?

A 
Extreme pH 
Shear forces
Air-water interfaces
Adsorption
Freezing, drying, rehydration
High pH/temperatures
High pressures
29
Q

What is preferential exclusion? Give and example

A

Protectant
Co-solute mainly out of the solvation shell of the protein
Excluded from unfolded state compared to the native state
Lower interaction with protein but not hydrophobic
= higher conc of co-solute in bulk than in solvation shel which draws water out and leaves the surface tight
e.g. Sucrose

30
Q

What is preferential binding? Give an example

A

Denaturant
Co-solute binds to surface of molecule within solvation shell
Interaction with backbone of protein unbinds the protein
e.g. H-bonding and guanidine hydrochloride

31
Q

Which stabilisers can be used with biologics?

A

Amino acids - decrease protein interactions, increae solubility and reduce viscosity
Polymers - exclusion and hydration
Polyols - accumulate in hydrophobic regions
Salts - exclusion/hydration
Surfactants - interfaces

32
Q

Which methods are used to stabilise biologics?

A

Acylation - with fatty acid to increase affinity to serum albumin and make longer acting insulin, glucagon and interferon
PEGylation - reduce plasma clearance rate and make admin less frequent
Surface engineering - modification of AA sequence to remove hotspot likely to cause aggregation

33
Q

What are the two types of studies used for determining shelf life during long term stability?

A

Accelerated studies

Stress studies

34
Q

Which temperatures are used for long term stability testing?

A

<20 +/- 5
5 +/- 3
25 +/-2 (60% RH) or 30 +/- 2 (65% RH)

35
Q

Which temperatures are used for accelerated stability testing?

A

5 +/-3 or 25 +/- 2 (60% RH)
25 +/- 2 (75% RH)
40 +/- 2 (75% RH)

36
Q

If shelf life is less than a year, what are the study intervals?

A

Monthly for the first 3 months then every 3 months

37
Q

If shelf life is more than a year, what are the study intervals?

A

Every 3 months during first year then every 6 months in second year then every year

38
Q

What are the problems with freezing biologics?

A

Cold denaturation - change pH, ionisation, solubility, H-bond energies
Repeated freezing and thawing = aggregation (nucleation points at ice-water interfaces)
Need cytoprotection by sugars, polyhydric alcohols and AA’s

39
Q

What happens during lyophilisation?

A

More long term stability
Reversible conformational changes which make prone to aggregation (+when reconstituted)
Reactions and denaturisation still occur (keep refrigerated and hydroscopic)

40
Q

What happens during freezing?

A

Ice crystals form and solutes get crystallised

  1. product frozen in vial
  2. put under vaccum below triple point of water
  3. dry heat causing ice to sublime

Pharmacy Y3S1 (14 decks)

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