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Pharmaceutics > Biologics > Flashcards

Biologics Flashcards

1
Q

WHat are some of the reasons for the higher success rate of biologics compared to small molecule drugs?

A
  • versatility (can replace diseased tissue)
  • specific binding (smaller drugs can bind to more sites, causing toxicity etc)
  • blood levels/duration of action (fast elimination of small molecules, mAbs have longer circulation times)
  • DDIs (lower/don’t happen due to targets)
  • immunogenic effects (high risk for therapeutic proteins, addressed by humanisation of tissues)
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2
Q

What are biosimilars?

A

generic equivalent - similar structure (NOT the same) - too many atoms to track. process may be different

cannot be treated the same as a small molecule generic

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3
Q

What is bioequivalence?

A

Not the same as where two small molecules are equally bioavailable

management of risk is crucial in demonstrating bioequivalence

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4
Q

What are the roles of amino acid side chains in proteins?

A

hydrophobicity, charge, polarity

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5
Q

What are the effect of hydrophobic side chains on proteins?

A

Mainly internal in globular proteins, external in membrane proteins

In globular proteins, the internal hyrodphobic chains are very stable, but when the protein starts to unfold they lead to aggregation and colloidal instability

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6
Q

Examples of the hydrophobic amino acid side chain?

A

Aliphatic: Ala, Val, Leu, Ile (and Gly)
Aromatic: Phe, Trp, Tyr

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7
Q

How do charged side chains affect proteins?

A

salt bridges - ionic bonds form

Asp and Glu have COOH
Lys and Arg have NH2

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8
Q

How do polar side chains affect proteins?

A

get hydrogen bonding

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9
Q

Examples of amino acids with polar side chains?

A

-OH : Ser, Thr, Tyr

C=O : Asp, Glu, Asn, Gln

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10
Q

How does protein aggregation occur?

A

Native protein has three outcomes: surface adsorbed (to container, can change shape), intermediate unfolding, or interaction with another molecule of the protein.

Intermediate can either return to native, or fully denature

Aggregates can form when denatured proteins interact, and if two molecules interact too closely

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11
Q

How do hydrophobic groups interact and cause aggregation?

A

Hydrophobic side chains are usually internal. When these become exposed, water is repelled, so want to bind with the other nearby hydrophobic groups

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12
Q

How can chemical degradation of proteins occur?

A

usually from exposure of side chains leading to unwanted interactions, e.g. via oxidation, hydrolysis, deamidation

there are so many possible conformations of proteins

exposure of hydrophobic groups, cysteine residues (-SH) or formation of disulphide bridges

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13
Q

What factors can influence instability and conformational changes?

A
  • pH extremes
  • shear forces
  • air/water interfaces (from agitation, stirring, shaking etc)
  • adsorption to solid surfaces
  • freezing, drying, rehydrating
  • elevated temp and pressure
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14
Q

What can be used to stabilise proteins?

A
  • amino acids
  • polymers
  • polyols
  • salts
  • surfactants
  • anti-oxidants
  • preservatives
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15
Q

How do added amino acids stabilise proteins?

A

preferential hydration and exclusion, decrease protein-protein interactions (interacts with side chains instead of two side chains interacting), increase solubility and reduce viscosity

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16
Q

How do added polymers stabilise proteins?

A

competitive adsorption to surfaces and interfaces, steric exclusion (form protective ‘layers’ around proteins)

preferential exclusion and hydration

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17
Q

How do added salts stabilise proteins?

A

preferential binding with ionic groups etc

interaction with protein bound water

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18
Q

How do added polyols stabilise proteins?

A

preferential exclusion, accumulation in hydrophobic regions

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19
Q

How do added surfactants stabilise proteins?

A

competitive adsorption at interfaces, reduce denaturation at air/water interfaces, also interfere with ice/water interfaces on freezing

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20
Q

How do added anti-oxidants stabilise proteins?

A

sacrificial to protect the protein, including in the discharge leak testing of finished vials - esp. liquid formulations

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21
Q

How are preservatives used in protein formulations?

A

often none in single use preparations as not needed, required if multi-use

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22
Q

What can be modified in the protein structure to improve stability?

A
  • acylation
  • PEGylation
  • surface engineering
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23
Q

How does acylation help stabilise proteins?

A

acylation with fatty acids to increase affinity to serum albumin - used to produce longer acting insulin, glucagon and interferon

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24
Q

How does PEGylation help stabilise proteins?

A

reduce rate of plasma clearance for less frequent administration (though some PEGylated proteins are less active, like antibodies)

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25
Q

What is PEGylation?

A

polyethyleneglycol polymers being put onto proteins - prevents the immune system destroying them (hydrophilic and uncharged, prevents recognition by immune system)

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26
Q

How does surface engineering help stabilise proteins?

A

removal of sites on the protein that are likely to cause aggregation

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27
Q

What is surface engineering of proteins?

A

Altering the DNA sequence to change the sequence of the protein, use of software to identify parts of the structure that can be changed

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28
Q

Importance of preferential interaction and exclusion?

A

Denaturants: interaction with the protein backbone, more interaction as the protein unfolds so can encourage denaturation (not good)

Protectants: interact with water more strongly, exclusion from protein structure. higher exclusion when protein unfolding, so encourages it to re-fold

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29
Q

What is preferential hydration?

A

interacts with water preferentially (over the protein)

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30
Q

How does preferential exclusion work?

A

a thermodynamic mechanism

degree of exclusion (and therefore the chemical potential) are directly proportional to the surface area of protein exposed to solvent.

the system favours the lowest chemical potential, so the smallest surface area of protein exposed. denatured proteins have highest SA so this is minimised when these excipients are added

31
Q

What is the double funnel model?

A

native state and aggregated proteins have a lower energy (more favourable) than the intermediate unfolded. bit like activation energy, once past a certain point of unfolding its more favourable to go to aggregation than to return to unfolded (but it takes sufficient energy to get from native to unfolding intermediate)

stabilising excipients increase the energy input required to reach the intermediate, so less likely to get aggregates

32
Q

Effect of low temperatures on proteins?

A

extends shelf life, cold denaturation can be reversible.

solvent properties change at lower temps, e.g. hydrogen bonds, diffusion rates, acid/base ionisation etc.

33
Q

Effect of freezing on proteins?

A

provides a lower temperature, but the repeated freezing and thawing process can increase aggregation - pH/conc changes, nucleation points for aggregation on ice/water interfaces

34
Q

What is cryoprotection?

A

sugars, polyhydric alcohols, ogliosaccharides, amino acids

mainly work by preferential exclusion, and lowering cold denaturation temperature, stabilising osmotic stress (surfactants protect against the interfaces)

35
Q

Effect of speed of freezing on the ice crystals?

A

slow cooling gives large crystals, fast cooling gives much smaller ones

36
Q

How does freeing encourage aggregation?

A

As water is removed from the solution into the ice, the proteins are more concentrated in the remaining solution, so closer together and more interactions

37
Q

Importance of mixing proteins after freezing?

A

The freeze-concentration effects will often remain after thawing if frozen vials not mixed

38
Q

What is lyophilisation?

A

Freeze drying - remove water. these proteins usually have a longer shelf life than solutions

39
Q

What are the issues associated with lyophilisation?

A

Proteins undergo reversible changes during the transition, otherwise buried sites get exposed so prone to aggregation
Freeze-concentration can still occur

similar things occur during reconstitution

40
Q

Why must lyophilised proteins still be kept refrigerated?

A

reactions still occur, so keeping cool reduces rate of this.

also keep sealed to avoid vapour absorption

41
Q

What is a eutectic mixture?

A

A mixture of two or more substances which melts at the lowest freezing point of any mixture of the components. This temperature is the eutectic point. The liquid melt has the same composition as the solid.

42
Q

How does lyophilisation work?

A
  • the solution is frozen (eutectic mixes freeze at lower temperatures than water)
  • water separates into ice crystals when the mix is frozen
  • pressure is reduced so solid goes straight to vapour and water is removed from the system
43
Q

How is accelerated stability testing done in proteins?

A

want to see the stability of the protein at normal temperatures, so increase the pressure at these to see how they respond - as high temps cause denaturation under a different mechanism so can’t really extrapolate data

44
Q

What is an example of how stability in proteins is measured in stability testing?

A

Measuring light obscurity as the pressure changes

45
Q

What is the protein structure of insulin?

A

monomer is 51 amino acids, naturally exists in a heximeric structure of six insuln monomers which adopts the structure of a globular protein

two Zn ions in centre, and histidine side chains hold insulin monomers to these

46
Q

How has insulin been modified to produce faster acting insulins for injection?

A

mutation of the amino acid sequence to disrupt assembly - converted to dimeric and monomeric structures.

This leads to faster diffusion and transport by faster action on sc administration, faster absorption across membranes (due to size) etc

47
Q

What are some of the mechanisms of changing the protein assembly?

A

Impairing dimerisation, charge repulsion at dimer interface, decreased zinc association

e.g. addition of protamine (highly cationic) aggregates insulin so ccan convert fast acting to internediate

48
Q

How can albumin binding be exploited to increase duration of action of insulin?

A

modified fatty acid side chain to increase the hexamer stability - leads to a long circulating depot of albumin bound insulin
Levemir

49
Q

How does significant variability arise in insulins?

A

Less variability with shorter acting insulins due to prandial administration
Early intermediate insulins formulated with protamine to create crystals, so speed of dissociation (and therefore absorption) can vary largely day to day

50
Q

How have insulins been advanced to reduced variability for patients?

A

Detemir has fatty acid modification which reversibly stabilises hexamers

51
Q

What modifications produced insulin glargine to increase duration? How is variability kept to a minimum?

A

changing isoelectric point from 5 to 7 by extending B chain with two arginines. precipitates on administration to blood, ezymes in the body remove these so slowly becomes active and has longer duration

variability is high as it relies on dissolution of isoelectric precipitates. care required with consistency of admin - same site, needle technique, time of day etc

52
Q

How are monoclonal antibodies produced?

A

B-cells are selected that produce the desired antibody, but must be fused with myeloma cells to produce a hybridoma (in order to grow them, B cells don’t grow well on their own)

often uses robots

53
Q

What are the types of antibodies used for mAbs?

A

Mouse, chimeirc, humanised, human

54
Q

Why were mouse antibodies used, and what are the problems associated?

A

Human hybridomas are difficult

Mouse antibodies different to human ones, so produce immunogenic response and rapid clearance

55
Q

What are chimeric antibodies?

A

Mostly human, but the variable region is still from mice. produced via recombinant DNA. less immune response when administered as proteins are mostly human

56
Q

What are monoclonal antibodies?

A

proteins produced by the B lymphocytes of the immune system in response to foreign proteins

57
Q

What are humanised antibodies?

A

Modified sequences to make antibodies identical to human ones, with a small non-human binding loop (complimentarity determining region)

58
Q

How are fully human antibodies made for mAbs?

A

IgG genes in a mouse replaces with the human genes, then onwards as normal

59
Q

How to monoclonal antibodies distribute once administered?

A

mostly within the central compartment (extracellular fluid) - penetration into cells is limited by molecular weight and hydrophilicity (except receptor mediated endo/transcytosis)

60
Q

What is the volume of distribution of mAbs?

A

0.1L/kg - approx equal to extracellular fluid volume

61
Q

What are the main mechanisms by which mAbs are cleared?

A
  • renal clearance (smaller molecules)

- proteolytic catabolism after receptor mediated endocytosis in the cells of the reticulo-endothelial system)

62
Q

Which parts of the body are in the reticulo-endothelial system?

A
  • microglial cells in the brain
  • alveolar macrophages
  • tissue macrophages (histiocytes) in connective tissues
  • Kupffer’s cells in liver
  • macrophages in lymph nodes and spleen
  • glomerular mesangial cells in kidney
63
Q

What is measured in the body before administering the next dose of mAbs?

A

the trough level

blood concentrations are spiky, but often over long periods (100+ days between doses)

64
Q

How are mAbs recirculated?

A

Brambell receptor (FcRn) - binds to Fc tail

65
Q

What is the FcRn receptor?

A

important for Ig homeostasis, and proteins like albumin

mostly expressed in vascular endothelial cells, or RES

saturates at high IgG levels, so creates an inverse relationship between dose administered and half life (half life also reduced in diseases where IgG is chronically high)

66
Q

How does FcRn recirculation work?

A

Avoids degradation in the lysosomes if antibodies are bound to FcRn

67
Q

How does loss of response to mAbs occur?

A

Anti-drug antibodies

68
Q

What effect do binding ADAs have?

A

Pharmacokinetic

Form immune complexes with the drug, so increased clearance rates. higher doses then required

  • have an indirect pharmacodynamic effect as there is a lower response
  • antibodies against the Fc region may reduce FcRn recycling and further clearance
69
Q

What effect do neutralising ADAs have?

A

have higher affinity, directly interfere with activity of the drug by binding to epitopes within or near to the active site (of the drug). similar PK effect, which is often greater than the pharmacodynamic.

70
Q

What factors can affect the efficacy of mAbs?

A

product related: homology to endogeneous proteins, non-glycosylated are more likely to be immunogenic, peptide affinity for antigen presenting proteins

non-product related: genetics, underlying disease (e.g. chronic inflammation), other medications, dosage

71
Q

How can antibody fragments be produced?

A

proteolytic enzymes and reducing agents

smaller ones can also be produced using recombinant DNA technology and fusing them with other effect proteins (use the antibody for the binding, protein for effect)

72
Q

What are antibody fragments used for?

A

imaging

lack the Fc, so no interaction with immune system and no FcRn recirculation

73
Q

What is Fc fragment engineering?

A

maintains Fc interaction with immune system and recycling - uses just the FcRn binding site and then antigen binding region etc

Pharmaceutics (5 decks)

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