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PHS3101 > Lecture 10: Protein biologics > Flashcards

Lecture 10: Protein biologics Flashcards

1
Q

Define biopharmaceutical.

A

A biological macromolecule or cellular component used as a pharmaceutical

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2
Q

What are the advantages of biopharmaceuticals?

A
  1. High specificity: very few side effects
  2. High potency: need a small amt to achieve therapeutic effect
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3
Q

What is the disadvantages of biopharmaceuticals?

A

High costs

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4
Q

What are biologics, biosimilars and biobetters?

A
  1. Biologics: (“biological products” — protein, sugar, DNA
  2. Biosimilar: biopharmaceutical with active properties similar to those of a previously licensed biopharmaceutical
  3. Biobetter: biopharmaceutical with active properties better than those of a previously licensed biopharmaceutical
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5
Q

How do we classify proteins, peptides, oligopeptides and polypeptides?

A
  1. Protein — > more than 50 amino acids.
  2. Peptide — > 2 to 50 amino acids.
  3. Oligopeptide — > 2 to 20 amino acids
  4. Polypeptide — > more than 20 amino acids
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6
Q

Describe the general structure of amino acids.

A

An amino acid consists of a carbon atom attached to an amino group, a carboxyl group, a hydrogen atom, and a side chain. The side chain varies between different amino acids, and determines the properties of the amino acid.

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7
Q

What is the primary sequence of a protein?

A

The amino acid sequence of the protein.
The amino acid residues are joined to each other by amine (peptide) bonds
They have the N and C terminus —-> free amine and carboxyl group respectively

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8
Q

What is the secondary structure of a protein, and what are the types of structures?

A

The secondary structure refers to the local folded structures that form within a polypeptide due to interactions (e.g. hydrogen bonds) between atoms of the backbone
Main types: α helix, β sheet, random coil

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9
Q

What is the tertiary sequence of a protein?

A

It is the overall three-dimensional arrangement of its polypeptide chain in space

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10
Q

What is the quaternary structure of a protein?

A

It refers to the spatial arrangement of subunits that come together to form the protein.

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11
Q

List the general properties of a globular protein.

A

Approximately spherical
Complex 3D structure with helixes and sheets
Soluble in water because the hydrophobic AA are enclosed in the interior and the hydrophilic AA are exposed on the protein surface and interact with water

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12
Q

What are some functions of globular proteins?

A

Cell signalling
Catalysis
Transport
Immunity

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13
Q

What is the isoelectric point of a protein and what is it dependent on?

A

The pH at which the protein molecule carries no electrical charge and it is dependent on the primary sequence of the protein.

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14
Q

What do we use to measure the charge on the protein?

A

Zeta potential

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15
Q

How does the pH and pI tell you the charge on the protein molecule?

A

pH < pI: positively charged
pH = pI: neutral
pH > pI: negatively charged

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16
Q

At what zeta potential are the protein molecules stable and why?

A

Magnitude 30mV and above
They have strong enough charges to repel each other and not aggregate.

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17
Q

How can the zeta potential be influenced by the conc of electrolytes in the solution?

A

A higher salt content means lower repulsion because the ions from the solution shield (neutralise) the charges of the proteins so they don’t repel each other as much.

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18
Q

Does protein aggregation increase or decrease with greater protein conc?

A

Increase

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19
Q

What are the segments of a monoclonal antibody?

A
  1. Antigen binding fragment (Fab) : variable heavy (VH) segment, variable light (VL) segment
  2. Crystallisable fragment (Fc): responsible for mediating immual responses
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20
Q

What are some stability problems with proteins?

A
  1. Denaturation
  2. Aggregation/precipitation
  3. Adsorption to surfaces (like the packaging)
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21
Q

How can misfolded proteins cause aggregation?

A

Unfolded/misfolded proteins — > hydrophobic amino acids exposed on the surface — > more
than one protein molecule “stick” together (aggregate)

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22
Q

How can bubble formation lead to proetin instability?

A

the proteins tend to migrate to the interface (air is more hydrophobic) and they unfold

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23
Q

Explain the process of freeze drying.

A
  1. Freeze liquid sample
  2. Primary drying: sublimation of ice under high vacuum
  3. Secondary drying: Evaporation of water from amorphous matrix + Desorption of chemiabsorbed moisture in the apparently dried cake
  4. Material storage
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24
Q

During the cooling stage of freeze drying, is the solid formed crystalline or amorphous?

A

Both
Crystallizable components — > crystallize
Non-crystallizable components — > for an amorphous matrix with unfrozen water

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25
Q

In a water phase diagram, the point below which sublimation occurs is the_________?

A

Triple point

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26
Q

What is the x and y axis used in a water phase diagram?

A

y axis is pressure
x axis is temperature

27
Q

What is the temperature used for primary drying?

A

Temperature needs to be 2 – 3 degC below the glass transition temperature of maximally freeze-concentrated solution (Tg) or the collapse temperature (Tc)

28
Q

How do Tc and Tg differ?

A

Tc is within 1-2 deg C of Tg

29
Q

Why shouldnt you dry at temperatures above Tg or Tc?

A

Drying above Tg or Tc may result in cake collapse — > impact product quality attributes such as residual water content, reconstitution time, and protein stability

30
Q

During primary drying, why does the sublimation rate decrease over time?

A

As more water sublimates, the layer of dried material on the surface thickens, increasing resistance for vapor to pass through. This resistance makes it harder for vapor to escape, slowing the sublimation rate over time.

31
Q

How much moisture is removed during secondary drying?

A

Removing 5–10% of the total sample moisture

32
Q

How is the dried sample stored after freeze drying?

A

Under inert gas (like N2) in a sealed vial to prevent reabsorption of moisture.

33
Q

What are the advantages of freeze drying?

A
  1. Increased stability because moisture can facilitate degradative processes like hydrolysis
  2. Reduced sample volume which is easier for further processing (e.g. purification) and storage
  3. Simplified transport –> no need to use cold chain shippage for perishable items
  4. Removing volatile buffer/solvent components which makes it easier to reformulate the powder since there is no intereference from other solvents
34
Q

What are the drawbacks of freeze drying?

A
  1. When water freezes the remaining liquid phase becomes more concentrated with excipient salts and proteins. This may cause them to precipitate, which can lead to denaturation of porteins and hence loss of function.
  2. During the freezing stage, the buffer salts may crystallize out of solution, causing a change in the pH, which can cause precipitation and denaturation.
35
Q

List the Key considerations in freeze-drying protein.

A
  1. Buffers should be kept to minimal concentration
  2. Mixed buffers arent preffered because pH shifts may occur during freezing (if one of the components freezes out before others)
  3. Salt concentrations should be reduced to a minimum (in the 10s mM range) so that they dont cause instabilities in the protein
  4. Use stabilizers like hydrogen bond forming sugars (eg sucrose) –
    >when water evaporates, they will replace water and form hydrogen bonds with the proteins to stabilize them
  5. Protein should be of sufficient dry weight for the formation of robust lyophilized cake
36
Q

What are the preffered buffers for freeze drying?

A

histidine, citrate or Tris buffers are preferable

37
Q

What are the steps for spray drying?

A
  1. Atomization: Solution feed is sprayed by an
    atomizer into a drying chamber
  2. Dehydration: Evaporation of water from solution feed droplets, heat transfer from hot air to droplets (large surface area)
  3. Powder collection
38
Q

What does infliximab do?

A

Binds to soluble & transmembrane forms of tumour necrosis factor α and prevents it from binding to its receptors –> stops inflammatory cascade –> treats Crohn’s disease and rheumatoid arthritis

39
Q

What are the 2 dosage forms of infliximab?

A
  1. Liquid dosage form
  2. Freeze dried powder (solid dosage form)
40
Q

Why is sucrose added to antibody drug formulations?

A

It helps to stabilise the protein

41
Q

Why are polysaccharides added to antibody drug formulations?

A

Increases the Tg
This is beneficial because then you can do primary drying at a highertemperature and save time

42
Q

Explain the process of freeze drying for infliximab.

A
  1. Frozen to -40deg C and kept at that temp for 2 h
  2. Primary drying: increase temp to -35 deg C and 0.05mbar pressure for 96 h
  3. Secondary drying: increase temp to 25 deg C, 0.01mbar for 24h
  4. Close vials under vacuum and keep at 4deg C
43
Q

What are the 2 ways used to produce infliximab in solid form?

A
  1. freeze drying
  2. spray drying
44
Q

What are the ways used to analyse infliximab once its synthesized?

A
  1. Gas chromatography to analyse moisture content
  2. SDS PAGE analyse protein structural integrity
  3. ELISA to analyse bioactivity
  4. Size exclusion chromatography to analyse protein aggregation
45
Q

What is the recommended amount of moisture content for dried biologics?

A

less than 3%

46
Q

How will you know that an antibody’s structural integrity is intact usign SDS PAGE

A
  1. Non reduced conditions: should be 1 band corresponding to the MW of the complete protein
  2. Reduced conditions: should be 2 bands corresponding to the MW of the light and heavy chain fragments
47
Q

Which part of the gastrointestinal tract is a good delivery site for proteinaceous drugs and why?

A

Colon
due to limited proteolytic effect since no proteases are released here and it has a long residence time
(this is acheived thru enteric coating which prevents the drug from being released into the stomach)

48
Q

What is trypsin used for?

A

Used in labs
It breaks down proteins responsible for cell attachment by cutting peptides at the carboxyl side of arginine or lysine residues

49
Q

What are the methods used to analyse the structural integrity of trypsin after tableting?

A
  1. Nα - Benzoyl - L - arginine ethyl ester hydrochloride (BAEE) assay
  2. Fourier transform infrared spectroscopy (FTIR)
50
Q

What does the Nα - Benzoyl - L - arginine ethyl ester hydrochloride (BAEE) assay analyse?

A
  1. it analyses te enzymatic activity of trypsin
  2. can evaluate how compaction pressure may change enzyme activity
51
Q

how does compaction pressure affect enzyme activity?

A
  1. increasing compaction pressure decreases enzyme activity because when you apply high pressure there are regions of high temp generated which can cause denaturation
  2. after a certain compaction pressure, the graph starts to plateau
52
Q

How can we use FTIR to analyse the effect of compaction on the denaturation of trypsin?

A
  1. compare the ftir spectra of both unprocessed and compressed trypsin
  2. if there are slight changes in the spectra we can conclude that there wer slight changes in the secondary structure of trypsin
53
Q

What are the 2 aspects that need to be considered during design of experiments?

A
  1. Critical quality attribute
  2. Critical process parameter
54
Q

What is critical quality attribute?

A

A physial, chemical, biological or microbiological property or characteristic of materials or products that should be within an appropriate limit, range or distribution to ensure the desired product quality.

55
Q

What are some examples of critical quality attributes?

A

purity (chemical and chiral)
particle size
crystal morphology

56
Q

What is critical process parameter?

A

A process parameter whose varibility has an impact on a critical quality attribute and therefore should be monitored and controlled to ensure the process produces the desired quality.

57
Q

Give some examples of critical process parameter.

A

temperature
stirring rate

58
Q

Define design of experiments.

A

An approach where the controlled input factors of the process are systematically and purposefully varied in order to determine their effects on the responses.

59
Q

What are the goals of DoE (design of experiments)?

A
  1. Determine the most influential critical process parameters
  2. Identify optimum factor settings that can improve product performance and ensure that critical quality attributes (CQAs) meet specifications with minimal variation.
  3. Understand how the factors interact with one another
60
Q

What are the advantages of DoE over the conventional way of experimentation?

A

in DoE, multiple factors are varied which allows you to see how the factors interact with each other.
in regular experimentation, you only vary one factor at a time which can be very time consuming & inefficient
Advantages:
1. maximise knowledge with minimal use of resources
2. predicts process behavior within the defined design space.
3. allows for multiple response optimization since every process has multiple CQAs that need to be optimzed tgt
4. Makes the process more robust so its less sensitive to external uncontrollable influences
5. indentify and handle outliers so they dont affect the results

61
Q

Explain the factorial design of experiments.

A
  1. Used to study the effects of multiple factors
  2. Each factor is represented by letters A B C D…
  3. Each level for the factors is represented by numbers
  4. number of experiments = number of levels ^ number of factors
62
Q

Explain the mixture design of experiments.

A
  1. It is to characterize and optimise a formulation
  2. Component proportions add up to 100% so when you increase the proportion of one component you decrease the proportion of the other
  3. Simplex vs optimal designs
  4. Simplex designs are like a triangle shape, where each corner corresponds to 100% of one component and 0% of the others
  5. Optimal: if you have an upper limit for the components the design cannot be potrayed by a triangle design + you need computer aided experimental designs
63
Q

Explain mixture process design of experiments.

A
  1. It is used to investigate interactions between formulation and process variables
  2. It requires the construction of mixture designs at each factor setting, which needs a large number of experiments
64
Q

What is the advantage of spray drying over freeze drying ?

A
  1. After freeze drying, the cake needs to be broken up to form small particles, which reduces the yield + its difficult to control particle size distribution.
  2. In spray drying, the particles are formed directly

PHS3101 (9 decks)

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