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The Medicine 2 - Sem 2 > Formulation of biologics > Flashcards

Formulation of biologics Flashcards

1
Q

What are biopharmaceuticals?

A

Medicines which contain:

mAbs, ADC, interleukins, peptides, virus like particles

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2
Q

What do protein therapeutics with enzymatic or regulatory activity do?

A

replace a protein that is deficient or abnormal, augment an existing pathway, provide a novel function or activity

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3
Q

What is the purpose of protein therapeutics with special targeting?

A

Interfering with a molecular pathway or organism’s physiology & delivering other compounds or proteins

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4
Q

What are the uses of protein vaccines?

A

to protect against foreign agent , treat an immune disease and treating cancer

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5
Q

What are the 4 different parts in naming mAbs?

A
  1. Unique prefix 2. Prefix letters related to type of target 3. Prefix reflects source of variable chain 4. Suffix mab
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6
Q

How are biologics formulated?

A

either as a liquid formulation or lyophilized to be reconstituted before use

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7
Q

How are biologics administered?

A

subcutaneously or intravenously or IM

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8
Q

What type of conditions are mAbs mainly used for and what are some problems with them?

A

long term conditions and necessity of injecting yourself or being in hospital may lead to compliance issues and they have the potential to elicit immune responses

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9
Q

What are the advantages & disadvantages of the lyophilized form?

A

Adv- dose and injection volume is adjustable so can potentially be adapted to patient and developed as multi-use formulation. Disadv – more expensive to couple to a delivery device such as the dual chamber

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10
Q

What are the advantages & disadvantages of the liquid form?

A

Adv – more convenient to end user, better compliance, more accurate dose. Disadv - storage in aqueous media increases the probability of chemical degradation by hydrolysis, less stable than lyophilised form , shorter shelf life, physical stability more difficult to control as shaking, air/water interface are likely to lead to unfolding of the mAb and ultimately its aggregation

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11
Q

What excipients are used in liquid formulations?

A

buffer, salt & tonicity modifiers , surface active agents, antioxidants, protein stabilisers, Cryoprotectants & lyoprotectants

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12
Q

What are some examples of buffers and why are they added?

A

acetate, citrate, histidine or phosphate used because pKa close to pH of interest, mAbs have an isoelectric point of about 8 and having solutions at pH between 5.5-7 guarantees good solubility

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13
Q

Why is buffer conc kept low?

A

to adapt to physiological pH upon administration to patient

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14
Q

What kind of preparation does an IV injection require?

A

isotonic preparation

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15
Q

What kind of preparation does an IM or SC injection require?

A

may be able to handle hypertonic or hypotonic conditions as they are not directly administered to blood

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16
Q

Why are salts used?

A

to modify tonicity which is the ability of a solution to make water move into or out of a cell by osmosis. they are needed to control stability/conformation of protein

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17
Q

Disadvantage of NaCl

A

contributes to corrosion of steel during bioprocessing and can affect the viscosity of the solution

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18
Q

What happens when mAbs unfold?

A

expose hydrophobic core, then either they refold or thy form aggregates with other unfolded mAbs in order to minimise contact of hydrophobic core with water

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19
Q

Why is the production of aggregates not acceptable?

A

can reduce therapeutic index and result in immunogenic response in patient

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20
Q

What are the surfactants used and why?

A

polysorbates 80 or 20 , they reduce unfolding as they will migrate to the interface and occupy it better than the mAbs

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21
Q

Why does the concentration of surfactant have to be above cmc?

A

to guarantee that all interfaces are covered and unfolding mostly happens at interfaces especially air/water interphase

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22
Q

Why is the chemical stability of polysorbates important?

A

they may oxidise and the free radicals will lead to aggregation of the mAbs

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23
Q

Why are antioxidants added and what are is the most common ones?

A

EDTA to limit chemical degradation through oxidization caused by the presence of metal ions in solution

Metal ions may have different origins such as metallic parts in the different steps of bioprocessing or the container in the formulation steps.

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24
Q

What type of agent can also reverse oxidation?

A

reducing agents such as glutathione

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25
Q

What types of protein stabilisers are used and why?

A

sugars and amino acids, they are used to limit unfolding of proteins, work by preferential exclusion, they remain out of the sphere of hydration and stabilise protein by attracting water molecules making the protein more compact

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26
Q

Are chemical and physical degradation linked?

A

yes because chemical degradation may lead to physical but physical can occur without chemical

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27
Q

What is the difference between Cryoprotectants and lyoprotectants?

A

stabilization is also an issue for lyophilized proteins so cryo protect molecule during freezing as water crystals may cause aggregation & lyo protect during drying

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28
Q

What is a problem with using sugars as lyoprotectants?

A

susceptible to hydrolysis at low pH and hydrolysis of sucrose forms glucose and fructose at pH5 which leads to protein degradation

29
Q

What are the most common routes for chemical degradation?

A

oxidation, de-amidation, aspartic acid isomerization and crosslinking

30
Q

What happens at the Complementary Determining Region of an IgG?

A

where most AA sequences modified as the region binds to antigen

31
Q

During modification of AA, the conformation or binding ability of mAb may change, why?

A

novel AA may have a different polarity or charge so will change the repulsion or attractive forces of the molecule; it may be also the result of the bio synthesis process

32
Q

What is de-amidation dependent on?

A

the AA

33
Q

What may aspartic acid isomerization lead to?

A

the degradation of 1 of the 2 CDR which in Herceptin leads to loss of activity

34
Q

Which AA are most susceptible to oxidation?

A

Methionine, Tyrosine, Histidine, tryptophan and cysteine

35
Q

Oxidation of tryptophan and histidine occur how?

A

via metal catalysed reactions

36
Q

What is meant by physical degradation?

A

Conformation changes, aggregation or surface adsorption due to physical stresses such as temperature, shaking, pressure.

37
Q

Extreme pHs or temperatures can lead to what?

A

covalent changes and affect conformation

38
Q

What are some sources of conformational changes?

A

temp changes, ice formation due to freeze thaw, shear forces, changes in ions in solution, changes in protein-protein interactions

39
Q

What are the 2 categories of aggregates and why are they undesirable?

A

reversible & non reversible, alter pharmacokinetics, reduce activity of mAb and lead to severe immune responses

40
Q

What causes reversible aggregates?

A

self association of the proteins perhaps form formulation or delivery, can revert to monomeric form by altering condition

41
Q

What are soluble aggregates?

A

aggregates not visible in solution and cannot by removed by filtration

42
Q

What are visible aggregates and what is the limit?

A

those larger than 10 m, less than 5% in weight compared to expected weight of proteins

43
Q

Proteins are hydrophilic disperse systems and are subject to what types of forces?

A

attractive, repulsive and solvation

44
Q

Among the 4 IgG subtypes which is more prone to aggregation?

A

IgG2

45
Q

How are protein candidates tested to determine presence of aggregates?

A

can be shaken, frozen and thawed or heated

46
Q

How do companies test to see if a candidate is prone to aggregation?

A

accelerated stability testing

47
Q

What are leachables?

A

Chemical compounds released from the container closure system when in stress conditions in the solvent

48
Q

Give examples of leachables

A

metals, organics, volatile compounds

49
Q

What are extractables?

A

compounds that elute during normal storage or use conditions

50
Q

What type of infusion bag is preferred for mAbs?

A

Polyolefin PO better than PVC

51
Q

Why should you consider the volume of the infusion bag?

A

having bags too large may result in large volume of air which increases possibility of protein damage

52
Q

What is the beyond use date?

A

The time the compounded sterile preparation must be used to avoid loss of potency, contamination, and safety risks.

53
Q

What is the head space?

A

the air volume left in the syringe after filling the desired volume of protein solution

54
Q

Why should headspace be minimised when filling syringe?

A

Because the air/water interface is believed to be the main source of unfolding of proteins and subsequently of aggregates formation.

55
Q

What is important to remember when reconstituting in larger volumes to reduce the protein concentration?

A

it will also reduce excipient’s concentration e.g of surfactants and if it is not above cmc then proteins will adsorb at interface and micelles will disappear

56
Q

What is the difference between IV and SC formulations?

A

SC not delivered directly into blood, better compliance, higher protein concentration, small volume injected, slowly released in blood compartment which means lower frequency of administration

57
Q

What are the challenges for SC formulations?

A

With increased concentrations, distance between proteins decreases and protein-protein interactions are more likely, which may mean more unfolding and more aggregation

58
Q

How does an increase in conc of a hydrophilic disperse system affect viscosity and why is that a problem?

A

will increase viscosity which will affect syringeabiltiy as patient would need to be very strong to inject themselves and as the internal diameter of needle is very small it can cause pressure build up

59
Q

What is the recommended glide force?

A

30 N which corresponds to a max viscosity of 25 cP

60
Q

The viscosity of mAbs formulation varies depending on what?

A

AA and excipients used

61
Q

What are some common forms of biopharmaceuticals?

A

Therapeutic monoclonal antibodies (mAbs), Insulin, vaccines, peptides used in hospitals such as teicoplanin and vancomycin

62
Q

How do we derive molecules to be used in biologics?

A

Peptides can be synthesised

mAbs, ADC and virus like particles are produced using biological processes

63
Q

Which host cells do we use to produce biologics?

A

The current host cells for the production are E. Coli, yeasts or Chinese Hamster Ovary (CHO) and Human Embryonic Kidney (HEK) cells

Process is complex, long and repetitive so often done by robots

64
Q

Which 3 ways can we utilise monoclonal antibodies in the treatment of disease and which sort of things can they treat in each instance

A
  1. Protein therapeutics which have an enzymatic or regulatory activity. They are proteins that replace another protein which is deficient or abnormal. They augment an existing cellular pathway (upregulates) or provide a new function or activity to the target cell.
  2. Proteins with special targeting as they can interfere with a molecular pathway or the organisms’ physiology, or, deliver other compounds or protein (case of ADCs).
  3. Protein vaccines which can be used to protect against a deleterious foreign agent, treat an immune disease or cancer.
65
Q

Explain the naming of mAbs

A

4 parts to each name

Part 1 - a unique prefix chosen by the company

Part 2 - letters in second prefix related to type of target

Part 3 - Prefix that reflects the source of the variable chain

Part 4: mab

66
Q

What features make a biologic unique?

A

The IgG isotype, their target, how these are delivered i.e. IV, SC, the concentration of the mAb formulation, the buffer and its pH and the main excipients of the formulation

67
Q

Give examples of cryoprotectants and how they work

A

PEG, sucrose or trehalose. These protect as water crystals may induce aggregation. They do work in a similar manner as the previously mentioned excipients i.e. preferential exclusion.

68
Q

How are each of the following amino acids oxidised:

Methionine, Histidine, Tryptophan and Cysteine

A

Methionine oxidation is frequent in mAbs (Herceptin again when exposed to light at 27°C and above).

Histidine oxidation often happens via metal catalysed reactions (which can happen during the bioprocess or in the final formulation (release of metal ions from the container).

Tryptophan oxidation occurs also via metal catalysed reactions (e.g. Trp in CDR of palivisumab when exposed to UV light) however this is rare in mAbs.

Cysteine has been shown in several mAbs to result in the cross-linking of intermolecular disulphide bridges.

69
Q

How do companies determine whether their product will aggregate

A

By using accelerated stability testing to determine whether their candidate is prone to aggregation. Different concept to the one used for small molecules: protein candidates can be shaken, frozen and thawed or heated to determine the presence of aggregates. However, this is only indicative and compared against other previous candidate results for long term evaluation.

The Medicine 2 - Sem 2 (14 decks)

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