Product Testing: Stability

Why do we need to do stability testing?

• Pharmaceutical products deteriorate upon storage

• All products have a shelf life / expiry date, which you need to know as a pharmacist

How do we formulate medicines?

To minimise degradation

Possible routes of degradation

• Oxidation

• Photodegradation

• Heat

• Solvent degradation

• Acid and base catalysis

Temperature and degradation

• Increase in temperature = increase in rate of degradation

• Can refrigerate (2-8^oC) a product if unstable at room temp

• Or freeze (<-15^oC)

Temperature and degradation

What is the problem with freezing medicines?

1. If heat is used for thawing = degradation

2. As the product melts = we end up with interfaces = proteins stick to interfaces, unfold and lose activity

3. Some drugs e.g. amoxicillin less stable when frozen than in the fridge » as changes solubility

4. Biopharmaceuticals and vaccines can be degraded by freezing

Temperature and degradation

Where is the most risk of exposure to elevated temps

Transport and storage in vehicles

What is chemical degradation?

• Any process where covalent bonds are broken

• So chemical structure changes

What can undergo chemical degradation?

• Both the active drug AND excipients

• There may be risk of the API reacting with the excipients

What group of chemicals are most drugs?

What is the problem with this?

• Most drugs are esters or amides

• If water is present, these can hydrolyse to alcohols and carboxylic acids

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solvent / the dosage form

1. Can replace water with another solvent

• We can choose this solvent based on the dielectric constant (a measure of polarity) » influences how fast charged species react

• Need to consider toxicity of solvent and compatibility with the drug (ensure it doesn’t react with the drug)

2. Using a solid dosage form instead

• Solid dosage forms more stable than liquid dosage form » as in a solution, every single molecule of drug is surrounded by water

• But: reactions can occur in water absorbed onto the surface of the particle

  E.g. poorly stored aspirin tablets smell of acetic acid (vinegar)

3. Freeze-dried powders and then reconstituted just before use

• For drugs that are very unstable e.g. penicillin

4. Formulate as a suspension

• Suspensions more stable than solutions

• Drug molecules in the centre of the particle protected from water

• Drug molecules on the surface may react with water

Why should you not store medicines in the bathroom?

Steam = water adsorbs to the surface of particles = hydrolysis

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

Why is pH of the solution important?

• A catalyst speeds up a reaction without being consumed itself » so a small amount of catalyst can cause a lot of degradation

• H3O+ and OH- (from the dissociation of water) can catalyse hydrolysis processes

  » so the drug will degrade faster at acidic or basic pH values

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

Graph of pH and degradation rate

Specific acid hydrolysis: hydrolysis catalysed by presence of H+

Specific base hydrolysis: hydrolysis catalyse by presence of OH-

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

Why is this graph important?

• Tells us the pH range where degradation rate is slowest (uncatalysed hydrolysis)

• Tells us when we need to add a buffer

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

What is this curve showing?

• Many drugs ionise

• And the neutral and charged forms of the drug degrade at different rates » hence, there is a point of inflection

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

Specific vs General catalysis

Specific catalysis: H3O+ and OH- catalysing degradation

General catalysis: other species catalysing degradation

How can we prevent hydrolysis of drugs that contain esters and amides?

Changing the solution itself

Examples of general catalysis

• Ions in buffers

• E.g. phosphate or acetate buffer catalyses the hydrolysis of chloramphenicol = incompatible with the drug

• Can use borate buffer instead which does not catalyse degradation = compatible with the drug

Light and degradation

Light causes degradation

To protect from light:

• Keep medicine in a tinted glass container / opaque outer container e.g. cardboard

• To block out UV light (which causes degradation)

Oxygen and degradation

• Oxygen causes degradation

To protect from oxygen (oxidation):

• We can flush the containers with an inert gas e.g. N₂, Ar, CO2 » to expel all the air and oxygen from the container

• However:

  • hard to remove all oxygen

  • best used for single-use containers as with multi-use, opening and closing allows for gas exchange and oxygen to enter

Oxygen and degradation

When does oxidation occur the fastest?

So how can we further prevent oxidation?

1. Oxidation is accelerated at high pH

• So we can formulate at low pH

  » However, there is a trade off between minimising oxidation and minimising hydrolysis

2. Metal ions e.g. Cu2+ and Fe3+ catalyse oxidation

• These are present in trace amounts in all formulations

• However they are not used up so can cause a lot of degradation

• We can add a chelating agent, like EDTA, to bind to the metal ion and prevent catalytic activity

3. Add an an antioxidant

• Reacts with O2 and removes it from the formulation

• Stops free radical reactions

• E.g. ascorbic acid (water soluble)

• E.g. a-tocopherol (oil soluble)

EQ:

What degradation might this drug undergo?

• Hydrolysis

• Oxidation (of OH group to a ketone)

• Heat degradation

EQ:

What does the region marked * on the diagram correspond to?

Uncatalysed hydrolysis

Physical Stability

Problems of containers for liquid dosage forms?

• Sorption of drug to container » leads to loss of drug

• Shedding of particles from glass container » poor appearance, poor mouth-feel (affects adherence)

• Extraction of materials from container into liquid » toxicity, change of pH

• Evaporation of volatile components e.g. peppermint » poor taste

Physical Stability

2 types of sorption that can occur

Adsorption: particles of drug adhere to the walls of the container

Absorption: molecules of drug taken up inside the wall of the container

» both cause the loss of drug from the solution

Physical Stability

When is sorption more likely to occur?

• Non-polar molecules have a high affinity for plastics and rubbers

  e.g. Diazepam in solution sorbs to plastic packaging

• pH can affect sorption » unionised form of the drug is less polar so more sorption to plastics and rubbers

Physical Stability

How can we prevent problems to do with containers for liquid dosage forms?

• Shedding of glass particles » use a plastic container

• If drug is hydrophobic and will stick to walls of a plastic container (non-polar) » use a glass container (polar)

• Evaporation of volatile compounds » don’t use rubber caps/lids

Physical Stability

Physical instability of solutions

• Precipitation of the drug = loss of efficacy, inaccurate dosing

• Precipitation of its degradation products = poor appearance

Physical Stability

Physical instability of suspensions

• Caking

• Ostwald ripening » even if drug is poorly water soluble, little bits of the drug may dissolve and may precipitate out

» can lead to inaccurate dosing, poor appearance, grittiness

Physical Stability

Physical instability of emulsions

• Creaming » dispersed phase droplets accumulate at the top

• Cracking » emulsion breaks into 2 separate phases

» poor appearance (affects adherence)

• Reduction in viscosity

» difficult application

» non-homogenous product

» increased risk of creaming and cracking

Physical Stability

Effect of changes in physical property

• Compromises appearance » can reduce adherence

• Compromises efficacy » inaccurate dosing, less drug present

What do we mean by preformulation?

• Understanding the properties of the drug

• We do this using qualitative measurements of the drug’s susceptibility to oxidation, hydrolysis and light degradation

Stability Testing: Preformulation

How do we measure the drug’s susceptibility to oxidation?

• Heat solutions of the drug with / without oxygen flushing

• Then look at chemical structure of the drug for degradation

Stability Testing: Preformulation

How do we measure the drug’s susceptibility to hydrolysis?

Heat solutions of the drug in water, acid and base

Stability Testing: Preformulation

How do we measure the drug’s susceptibility to light degradation?

Shine artificial daylight lamps on the drug

Stability Testing: Preformulation

What other preformulation test do we do?

• Investigate stability of the drug in the solid state

• Keep the drug at high temp

• Add the drug with possible excipients

Stability Testing: Stress Testing

High temperatures

What does this involve?

• Storing the drug at high temperatures

• We then calculate the rate of degradation at RT using the Arrhenius equation:

  k = Ae^-Ea/RT

• You then plot a graph of k vs the different temperatures

• Calculate equation of the line

• Determine what the rate of reaction at room temperature should be

• Carry out with the drug alone then repeat with drug + excipients then with candidate formulations (exactly how the patient will take the drug)

Stability Testing: Stress Testing

High temperatures

Advantages

• Speeds up the degradation process instead of waiting for years For degradation to happen at room temp

• So we get results quickly

Stability Testing: Stress Testing

High temperatures

Limitations

1. Predicted shelf life at room temp is inaccurate

• Different reactions may occur at higher temperatures than room temperature

• Degradation products may themselves break down

  E.g. if trying to quantify the amount of degradation product (B) but the degradation product (B) breaks down (C), we will not be able to quantify the C as we are only looking for the B

2. Heating reduces moisture levels in solid products

• Underestimate rates of hydrolysis

3. The stability of the product must be studied in the actual storage conditions to be used

Stability Testing: Stress Testing

Temperature Cycling

What does this involve?

• Subjecting liquid products to freeze/thaw cycles (very high then very low temps)

  This can cause:

• Ageing and particle growth in suspensions,

• Cracking in emulsions

• Precipitation in solutions

Stability Testing: Stress Testing

Photostability Testing

What does this involve?

• Shine an artificial daylight lamp on pure drug

• Then repeat with formulated product

• Then use same packaging as will be used when drug is released to market

Stability Testing: Long Term Testing

What is long-term testing?

• Long-term studies performed under actual storage conditions

• After preformulation and stress testing, we should now have a formulation that we are confident is stable

• Long-term testing is used to validate this to show a regulator that the drug is ready to be released to market

Stability Testing: Long Term Testing

What do we do during long-term testing?

• Use worst case scenarios of temperature and humidity E.g. in particular regions of the world, how hot and how humid does it get

• Use packaging that is intended to be used in the market

• Samples are removed for 12 months

• We then assay for both the drugs and excipients

• Might also need pH tests, microbial tests and physical characteristic tests

Stability Testing: Long Term Testing

Requirements for long-term testing?

• Use same packaging for testing as will be used for the product

• Test at least 3 batches of the product

• Store liquid formulations inverted to ensure that the contents interact with the cap of container

• Sample every 3 months in year 1, every 6 months in year 2 and then annually until the end of the proposed shelf life

Stability Testing: Long Term Testing

How big are the samples used?

Batches of at least pilot scale

Stability Testing: Long Term Testing

What are we looking for when we sample every 3 months/6 months/annually?

Oral solutions, suspensions, emulsions:

• Precipitation

• Clarity (solutions)

• pH

• Viscosity

• Extractables

• Microbial contamination

Suspensions:

• Dispersibility

• Rheological properties

• Size and distribution of particles

Emulsion:

• Phase separation

• Size distribution of dispersed phase

Stability Testing: Long Term Testing

Criteria for results

• Degradation must leave >90% of stated dose left » use statistical tests

• Appearance, smell and taste must be acceptable

• Test at least 3 batches, calculate shelf lives and take the shortest shelf life

When do we NOT need to control humidity in stress testing / long term testing?

For aqueous products in glass containers

Purpose of pre-formulation and stress testing summary

Preformulation: tells us which degradation will pose the biggest problem

Stress testing: tells us the rate of degradation and quick information about the stability of the drug

EQ:

Explain the differences between the four global climactic zones. Why is it important to study product stability in all the zones where the product will be sold?

• The four zones differ in their average temperatures and relative humidities.

• It is important to study product stability in all the zones where a product will be sold in order to ensure that it is stable in that set of conditions: temperature and relative humidity (RH) will affect degradation rates and processes.

EQ:

What is caking, and why is it problematic in a pharmaceutical suspension?

• Caking arises when particles in a suspension sink very slowly

• This results in minimal liquid trapped within the particle

• So the particles are very difficult to disperse upon shaking

• This means most of the drug will be stuck at the bottom of the container

• Leading to inaccurate dosing

EQ:

Why do we test multiple batches of a product during long-term stability testing?

 

• There will be batch to batch variability - not all batches will be identical

• So we need to test several batches to ensure we have a good overall understanding of how the formulation will behave