Stability



Fig. 22.1
pH stability relationship for acetylsalicylic acid solution [11]



Hydrolysis is not only dependent on the pH (catalysis by H+ or OH ions), but may also be catalysed by certain ions, such as phosphate and acetate. In general, higher ion concentrations promote hydrolysis reactions [12]. In a buffered solution, hydrolysis thus depends on the pH, the type of buffer and the concentration of that buffer.

Hydrolysis rates increase by 2–3 times for every 10 °C temperature increase. Note that the pH of a buffered solution may shift when the temperature changes [13]. Heating of a solution may thus influence the hydrolysis rate both through temperature and pH. Changes in pH may also be important during freezing and freeze-drying. Gradual freezing of an aqueous solution results in the formation of ice crystals and a concentrated salt solution, in which hydrolysis rates can be increased due to a higher concentration or a pH shift, or both. Therefore, freezing should be done as fast as possible.

Borate buffers show a minimal pH shift when the temperature increases (e.g. during heating or sterilisation) or decreases (freezing) and the concentration that is required to achieve an isotonic solution is relatively low. These two factors contribute to this buffer’s suitability for ophthalmic solutions (see Sect. 10.​6.​1).

Hydrolysis of an ester can result in a substance with reduced aqueous solubility; possibly leading to precipitation even when relatively little degradation has occurred. An example is the hydrolysis of prednisolone disodium phosphate and the formation of the poorly soluble prednisolone.

Examples of substances that are prone to hydrolysis are: acetylsalicylic acid, ampicillin, barbiturates, chloramphenicol, chlordiazepoxide, cocaine, corticosteroid phosphate or succinate esters, proteins, folinic acid, indomethacin, local anaesthetics, paracetamol (acetaminophen), pilocarpine, tropa alkaloids (atropine, scopolamine), xylomethazoline and the antimicrobial preservatives methyl and propyl parahydroxybenzoate. In the field of oncology, melphalan and bendamustine hydrochloride are highly susceptable to hydrolysis with a shelf life of 1.5 h for melphalan and 3.5 h for bendamustine at room temperature.


Cocaine Eye Drops

The hydrolysis of cocaine strongly depends on pH and temperature. To minimise degradation, the pH should not exceed 5.5. Therefore, a boric acid – benzalkonium solution is suitable for the preparation of iso-osmotic solutions, with a pH of 4.5–5 (see Table 22.1).


Table 22.1
Cocaine Hydrochloride Eye Drops 5 % [14]





















Cocaine hydrochloride

5 g

Benzalkonium chloride

0.01 g

Boric acid

0.4 g

Disodium edetate

0.1 g

Water, purified

ad 100 mL

Cocaine may degrade into benzoylecgonine and subsequently into ecgonine and benzoic acid [15]. The degree of degradation that results from heating the solution has been investigated. The unheated solution (pH 5.0) contained 1.3 % benzoylecgonine, expressed as percentage of the cocaine content. After 30 min of heating the solution at 100 °C, the benzoylecgonine content had increased to 2.1 % and after 15 min at 121 °C to 2.7 %. Due to the weak buffer in the solution, the degradation resulted in a pH decrease to approximately 3.

The process of hydrolysis can be inhibited by using the right pH, by reducing the storage temperature, or by processing the active substance in the solid state. Solid state active substances and medicines should be stored under dry conditions to prevent hydrolysis.

The pH of a prednisolone oral solution (Table 22.2) is set at 7–7.5 to limit the hydrolysis of prednisolone disodium phosphate. After 12 months of storage, no degradation is measurable. However, hydrolysis of the preservative methyl parahydroxybenzoate limits the shelf life to 12 months, as the amount of preservative has then been decreased by 25 %.


Table 22.2
Prednisolone Oral Solution 1 mg/mL (as disodium phosphate) [16]

































Prednisolone sodium phosphate

0.146 g

Bananas flavouring (local standard)

0.1 g

Disodium edetate

0.1 g

Disodium phosphate dodecahydrate

1.9 g

Methyl parahydroxybenzoate

0.15 g

Sodium dihydrogen phosphate dihydrate

0.21 g

Sorbitol, liquid (crystallising)

25.8 g

Water, purified

77.5 g

Total

106.8 g (= 100 mL)



22.2.2 Oxidation and Reduction


Oxidation is a chemical reaction during which a substance loses (donates) electrons, whereas during reduction an electron is taken up (accepted) by the substance. Active substances are decomposed more often by oxidation than by reduction. Typical oxidation processes involve organic molecules that react with oxygen molecules that are dissolved in water or present in the air. The process usually consists of a cascade of reactions through the formation of free radicals, which are molecules or atoms that are highly reactive, due to the presence of one or more unpaired electrons. Free radicals exist as the superoxide anion (•O = O-), hydroxyl radicals (•OH) and carbon (chain) radicals (•R). Many oxidation reactions are catalysed by traces of heavy metals.

The mechanisms and kinetics of oxidation reactions are very complex, which makes it difficult to predict whether oxidation reactions of organic molecules will occur and how to prevent them from happening.

The oxidation reaction rate is usually not dependent on the concentration of the substance (see Sect. 22.5.7). Therefore, the content of low-dose preparations decreases relatively faster by oxidation than that of high-dose preparations. This is in contrast to the process of hydrolysis, during which the percentage of the substance that degrades is more or less constant per unit of time. Like all chemical reactions, oxidation proceeds more slowly at lower temperatures. However, oxygen molecules dissolve better in water and fats at lower temperatures, which implies that more oxygen will be present in the formulation to initiate oxidation reactions. The degree of oxidation of many substances in aqueous solutions is dependent on the degree of ionisation of the substance. Lowering the pH will lead to protonation of nitrogen atoms in amines and blocking of easily excited electrons in several organic molecules [17]. Those molecules will therefore be protected by a low pH. This is only the case for basic substances.

An example of a simple oxidation reaction is the conversion of Fe2+ into Fe3+. Examples of active substances that are prone to oxidation are acetylcystein, adrenaline, apomorphine, clioquinol, dithranol, dobutamine, ergotamine, hydroquinone, isoniazid, mesalazine, naloxone, neomycin, oxycodone, paracetamol, peptides, salbutamol, phenothiazine derivatives (promazine, promethazine, chlorpromazine), phenylephrine, physostigmine, tetracycline, tretinoin, the vitamins A and D, and the excipients: flavouring agents, fragrances and unsaturated fats (vegetable oils, suppository bases).

Reduction of active substances occurs more infrequently than oxidation. Some examples are:



  • The conversion of hydrogen peroxide into oxygen


  • The reduction of methylthionine (in the solution for injection) into its leuco-form, which may be the active form anyway


  • The formation of metallic silver in an aqueous solution of silver nitrate that is exposed to light and that contains a trace of an organic substance to prime the reaction

Oxidative reactions often proceed into polymerization reactions, resulting in large molecules of which the complex structures can be difficult to determine. A well-known pharmaceutical example is the brown discolouration of adrenaline solution.

Upon degradation, adrenaline solution first turns into pink, then red, and finally brown. Adrenaline is first oxidised into adrenochrome, which is consecutively oxidised into the fluorescent adrenolutin and brown melanin products [18]. The oxidation rates increase with increasing pH. The stability is the best at pH 3.2–3.6, by virtue of the relatively low reaction rate of the first step at this pH [19]. At pH 7.4, the rate of the second step is relatively high, whilst at pH 6.9, accumulation of adrenochrome occurs [18].

The raw material paracetamol may hydrolyse into aminophenol at high relative humidity. Subsequently, aminophenol is oxidised into toxic quinonimines and related substances [6]. This process causes a discolouration of the powder from white to pink, brown, and black. Discolouration of a paracetamol solution may imply that the raw material was stored under humid conditions. To be able to be aware of toxic degradation products, it is advised not to add a coloured flavouring agent, like caramel, to a paracetamol solution.

Oxidation processes can be inhibited by limiting the availability of oxygen and sometimes by the addition of antioxidants.


22.2.2.1 Limiting the Availability of Oxygen


An effective way to prevent oxidation is to remove oxygen from the water that is used in the formulation and to prevent the influx of oxygen after preparation. It is nearly impossible to completely remove oxygen from water, oil or fat. Preventing the influx of oxygen is only possible if the preparation is packed separately per dose. For injectable, oxygen sensitive substances, in ampoules, flushing with and packaging under an inert gas is a common and effective method to decrease the oxygen content. However, the transfer of a solution for injection from an ampoule into a syringe allows oxygen to dissolve in the water. Therefore, it is usually not possible to prepare the syringes far ahead when dealing with oxidisable active substances (for example at the Centralised Intra Venous Additives Service (CIVAS)).

In multidose containers, the ingress of oxygen can only be prevented marginally. For aluminium tubes, squeezing them and rolling them up helps to a certain extent. For bottles, limiting the empty volume in the top may increase the shelf life of the unopened container. By substituting a part of the water in an aqueous solution for a concentrated sugar solution, glycerol or propylene glycol, the solubility of oxygen is diminished, and thus oxidation. However, in many situations it is essential to add antioxidants, substances that reduce oxidation.


22.2.2.2 Antioxidants


Oxidation reactions can be inhibited by three types of antioxidants [20]:

1.

True antioxidants: these are thought to block chain reactions by reacting with free radicals. The agent (e.g. DL-alpha-tocopherol, butylhydroxytoluene) donates electrons and hydrogen atoms, which are accepted by free radicals, more easily than the active substance itself.

 

2.

Reducing agents: these have a lower redox potential than the active substance or excipient they are protecting. The agent (e.g. sodium metabisulfite, sodium formaldehydesulfoxylate, ascorbic acid) is oxidised more easily than the active substance.

 

3.

Antioxidants synergists: these enhance the effects of antioxidants. For instance by the binding of copper or iron ions, which catalyse the oxidation reaction. Usually, complexing agents are used for this purpose (e.g. disodium edetate, citric acid).

 

The mechanism of action of a substance that is oxidised more easily than the active substance can be explained by Nernst’s law:


$$ E={E}_0+\frac{0,059}{n} \log \frac{\left[ ox\right]}{\left[ red\right]} $$

(22.1)
in which E = redox potential

E 0  = standard redox potential

n = number of electrons in the redox equation

[ox] = concentration of substance in oxidised form

[red] = concentration of substance in reduced form

This equation can be used for both the active substance and the antioxidant. The active substance is protected when the redox potential of the antioxidant is several units lower than the redox potential of the active substance, which is the case when both the standard redox potential of the antioxidant is lower than that of the active substance and the antioxidant is mainly present in its reduced state. For a given oxygen load, protection against oxidation lasts longer when the initial concentration of the antioxidant is higher. Some active substances have a very low standard redox potential, which renders protection by the usual antioxidants ineffective. In these cases, elimination of oxygen is the only way to prevent oxidation.

The Nernst equation can only be applied when the redox potentials of both the active substance and the antioxidant are known.

Generally, the choice for an antioxidant or combination of antioxidants and the required concentration are determined experimentally. This choice is also dependent on the phase that should contain the antioxidant, either the water phase or the oil phase. The antioxidants that act via the first mechanism are fat-soluble; those that act via the second and third mechanism are water-soluble. Some examples follow showing the importance of actually testing the anticipated effect of an antioxidant.


Example 1. Blocking the Oxidation of Tretinoin

Butylhydroxytoluene decreases oxidation of tretinoin both in an ethanol/propylene glycol mixture (Table 22.3) and in cetomacrogol cream (Table 22.4).


Table 22.3
Tretinoin Cutaneous Solution 0.05 % [21]





















Tretinoin

0.05 g

Alcohol denaturated 95 % V/V (local standard)

40.4 g

Butylhydroxytoluene

0.05 g

Propylene glycol

52 g

Total

92.5 g (= 100 mL)



Table 22.4
Tretinoin Cream 0.05 % [22]





















Tretinoin

 0.05 g

Alcohol denaturated 95 % V/V (local standard)

12 g

Butylhydroxytoluene

 0.04 g

Cetomacrogol cream FNAa

88 g

Total

100 g


aCetomacrogol emulsifying wax (BP) 15 g, sorbic acid 200 mg, decyl oleate 20 g, sorbitol, liquid (crystallising) 4 g, water, purified 60,8 g. Total 100 g

On the contrary, a different antioxidant, DL alpha-tocopherol, increases oxidation in cetomacrogol cream. This phenomenon is caused by peroxides, which originate from DL-alpha-tocopherol oxidation in the oil phase. The peroxides generate free radicals that initiate the oxidation of tretinoin.


Example 2. Instability of Acetylcysteine

Acetylcysteine decomposes in aqueous solution by hydrolysis and oxidation. The oxidation into N,N-diacetylcystin is said to be predominant [23, 24]. Furthermore, hydrogen sulfide and other sulfur containing substances can be formed. The shelf life of the medicine is not only limited by the requirement of a maximum of 10 % decomposition of acetylcysteine, but also by the unpleasant smell of the sulfur-containing degradation products. By HPLC the effects of antioxidants on the formation of N,N-diacetylcystin in acetylcysteine eye drops (Table 22.5) could be determined.


Table 22.5
Acetylcysteine Eye Drops 5 % [25]





















Acetylcysteinum

5 g

Benzalkonium chloride

0.01 g

Disodium edetate

0.1 g

Trometamol

5.3 g

Water, purified

ad 100 mL

It appeared that ascorbic acid, sodium metabisulfite or sodium formaldehyde sulfoxylate had no effect or even increased the amount of N,N-diacetylcystin and potentiated the smell of sulfur containing substances.


Example 3. Dithranol Cream

Ascorbic acid has been added to a dithranol cream (Table 22.6) following research results with similar preparations [27, 28]. According to the literature [28] the combination of ascorbic acid and salicylic acid protects dithranol in a cetomacrogol cream better than salicylic acid alone. If ascorbic acid and salicylic acid are omitted, the degradation of dithranol amounts up to 40 % instead of 10 % within 1 month at room temperature.


Table 22.6
Dithranol Cream 0.05 % [26]





















Dithranol

0.05 g

Ascorbic acid

0.1 g

Salicylic acid (90)

  1 g

Lanette cream I FNAa

98.85 g

Total

100 g


aSee Table 12.​34

The concentration of the antioxidant is related to the total volume of the preparation instead of the active substance content, since the volume determines the oxygen content. When a preparation that contains an antioxidant has to be diluted, caution should be exercised to ensure that the end concentration of the antioxidant is not too low to be effective.

‘True’ antioxidants (type 1, see above) and reductants (type 2, see above) are used up during the shelf life, which implies that their content is much lower at the end of the shelf life than when the preparation was released.

Other aspects of antioxidants should be considered:



  • Sodium metabisulfite reacts with oxygen with the formation of sulfate, which results in a pH drop


  • Sodium metabisulfite may bind to certain active substances, such as prednisolone disodium phosphate and adrenaline tartrate

Adrenaline reacts in equimolar proportions with sodium metabisulfite and is thereby inactivated [29a]. Because of the equimolarity of this reaction, changing the concentration of either adrenaline or metabisulfite may change the shelf life of the preparation quite drastically. It is supposed that metabisulfite at first reacts with oxygen. Therefore, the availability of any metabisulfite that is left for the reaction with adrenaline also depends on the amount of oxygen in the preparation. Moreover, this reaction is influenced by the pH, with a maximum rate at pH 2–3 [30].

To add to the complexity of the preparation, bisulfite has a negative effect on the stability of adrenaline in presence of light. Therefore, it may be necessary to avoid the use of bisulfite if the solution is exposed to light during the administration, especially during continuous infusion [31].

Oxidation of ascorbic acid causes a yellow discolouration. A preparation that contains this substance can turn yellow, whilst the active substance may still be unaffected. Therefore, masking the discolouration of ascorbic acid in a promethazine syrup with caramel is defendable.

Many of the antioxidants and complexing agents that are used in pharmaceutical preparations are also used in food products. Since food can be taken daily, limits have been established for these substances. These limits are called Accepted Daily Intake (ADI) in the EU and Recommended Dietary Allowance (RDA) in the US. In many countries, these limits also apply to pharmaceuticals. Table 22.7 summarises the antioxidants, their European identification number, their ADI, and their usual concentrations in pharmaceutical preparations according to [32, 33].


Table 22.7
Antioxidants with their E-number, ADI and Usual Concentrations












































Antioxidant

E-number

ADI (mg/kg body weight)

Usual concentration (%)

Ascorbic acid

E 300

No limit

0.01–0.5

Butylhydroxyanisole (BHA)

E 320

0.5

0.01–0.02, but less in i.v. injections

Butylhydroxytoluene (BHT)

E 321

0.3

0.01–0.02, but less in i.v. injections

Sodium metabisulfite (sodium pyrosulfite, sodium hydrogen sulfite)

E 223

0.7

0.01–0.2

Sodium formaldehydesulfoxylate

a
 
0.1

DL-alpha-tocopherol

E 307

2

0.05–0.1


aNot allowed in food


22.2.3 Isomerisation


Isomerisation is the transition of one isomer of a molecule into another isomer. During this transition, the configuration (spatial arrangement of molecular groups) around an asymmetric carbon atom changes. Two types of isomerisation can be distinguished: racemisation and epimerisation. In the case of racemisation the molecule has one centre of asymmetry. In case of epimerisation the molecule has two or more centres of asymmetry, of which one is involved in isomerisation. Isomerisation is a reversible process; eventually equilibrium between both configurations is established. A solution of one of the configurations is optically active. Racemisation results in a racemic mixture without optical activity. On the contrary, for epimerisation the optical activity remains in equilibrium. In many cases pH influences the rate of isomerisation.

A well-known example of isomerisation is the conversion of ergotamine into ergotaminine and, if the other carbon atom also is affected, in aci-ergotamine. Other active substances that degrade by isomerisation are adrenaline, colecalciferol, ergometrine, pilocarpine and tetracycline.

Racemisation of L-adrenaline is accelerated by light and acid. At pH 4.5, racemisation is minimal. Temperature has a large influence: Q10 = 2.8 (see Sect. 22.5.7). The leftward rotating isoform is 15–20 times more active than the rightward rotating isoform, so racemisation results in loss of activity.


22.2.4 Photolysis


Photolysis is chemical degradation facilitated by light. The standard reference on this topic is the book Photostability of drugs and drug formulations [17].

Light is a form of energy. When a molecule absorbs this energy, its energetic state is increased and in some cases, ionisation can occur. The molecule may return to its original stable state, or it may degrade. In principle, all types of degradation can follow the absorption of light, but most often oxidation occurs. This combination of events is called photo-oxidation. Light can also be absorbed by excipients that subsequently transfer a different kind of energy to the active substance, which in turn may degrade. This process is called photo-sensitisation. Examples of excipients that can act as sensitizers are the antioxidant metabisulfite and furfural, which is the degradation product of glucose that is present in trace amounts in glucose 5 % infusion solutions [34].

The light spectra that can be absorbed by a substance can be determined from the substance’s absorption spectrum. Coloured active substances absorb visible light; the light spectrum that is absorbed is complementary to the colour of the active substance. White organic substances absorb UV light, corresponding to their UV spectrum. The presence of conjugated double bonds or an aromatic ring are an indication of the ability of the substance to absorb UV light and thereby be susceptible to photolysis.

The most reactive part of the spectrum is UV-B (280–320 nm), which is responsible for most direct photo-chemical degradation reactions of active substances. UV-A (320–400 nm) is more likely to induce photo-sensitisation. Infrared light is only relevant in the sense that it transfers heat, and may thus increase the reaction rate of degradation processes. Sunlight has a very broad light spectrum. Glass blocks most UV-B light present in sunlight, but still enough may pass through to induce degradation of active substances in glass containers, for example in a container that is kept on the window sill, or in an infusion bottle hanging from the bed of a hospitalised patient. Fluorescent light, in some countries known as tube luminescent (TL-)light, contains a fraction of UV light.

Some well-known photochemical degradation reactions of active substances are:



  • The formation of p-nitrobenzaldehyde from chloramphenicol


  • The formation of lumi-derivatives from ergotamine


  • The separation of a side chain from methotrexate

Also the oxidations of chlorpromazine, cyanocobalamine, dithranol, nifedipine, tretinoin and primaquine are accelerated by light. In oncology, carmustine, fotemustine and dacarbazine are examples of active substances that are very sensitive to light. Moreover, the degradation products of dacarbazine are suspected of being toxic to the patient.

Photolysis can be prevented or reduced in the following ways:



  • Prevent the light from reaching the medicine product.


  • Exclude oxygen or add radical scavengers or UV absorbing substances.


  • Reduce the exposure to light during manufacturing.


  • Administer the medicine in subdued light or cover the skin with clothes after administration of a dermal medicine.

A coating or protective secondary packaging can be used to prevent light from reaching the active substance.

A tablet coating can protect the core against photolysis if this coating is not light transmissive, for example because it contains pigments. Suitable non-light transmissive secondary packagings are carton boxes and aluminium tubes. Brown glass may provide enough protection for its contents, but not always.

For coloured, light-protective glass, the Ph. Eur. contains requirements on the transmittance of light with a wavelength of 290–450 nm. These requirements provide a certain standardisation of coloured glass, but they do not guarantee that coloured glass protects against all kinds of photolysis. For example, phytomenadione in oily solution, colchicine tablets and tretinoin solution are not protected sufficiently by brown glass or opaque plastics. In addition to the brown bottles in which they are packaged, these products should be stored in the dark.

The disadvantage of an overwrap is that the patient or nurse may want to take it off and not put it back on, which may result in problems in the following situations:



  • For parenteral medicines that should be prepared for administration and subsequently administered, wrapping the medicine conflicts with the desire to continuously check the solution for clarity and volume. If a light-sensitive active substance is to be administered, the duration of the administration should be limited. A nicardipine intravenous infusion can be exposed to light for up to 8 h, which is sufficient time for administration [35]. A furosemide intravenous infusion can even be exposed to light for up to 24 h [36].


  • Repackaging of solids in automated medicine dispensing systems should be done in such a way that the light protective capacity of the original container is met. In practice however, the new packaging material is often more transmissive to light than the original packaging material. Still, it is generally assumed that in most cases, the shelf life in the dispensing bag is long enough to cover the storage period of such bags, which is usually 1 week. An example of an active substance that requires specific attention in these situations is nifedipine.

Oxygen is usually involved in many photochemical reactions, thus the elimination of this substance is an effective means of protection against these reactions in many cases. Radical scavengers can be used to reduce the reactivity (“quench”) of radicals or oxygen singlets. Ascorbic acid, DL-alpha-tocopherol and butylhydroxytoluene are used for this purpose. Also lactose, mannitol, saccharose, starch and povidone can have a quenching effect [17b]. Another strategy is to add UV-absorbing substances, such as p-aminobenzoic acid, benzophenones or vanillin, to a light sensitive active substance [17c].

Some active substances are so sensitive to light that light must be excluded to the highest possible extent during preparation. In practice, this means that the preparation should not be done in the vicinity of a window and that artificial lights should be switched off. Examples of pharmaceutical preparations for which this may be necessary are dithranol cream, phytomenadione oral solution and tretinoin solution.

The final question that should be raised is would it be necessary to protect light sensitive active substances if the patient uses the preparations on his skin or in the eye. The answer is that it depends on the rate of degradation and on the toxicity of the degradation products.

As an example, the photodegradation products of chloramphenicol (nitroso-compounds and paranitrobenzaldehyde) are considered carcinogenic. These products will be formed in vitro and in vivo and can reach the bone marrow in rats [37]. Chronic use of chloramphenicol has been connected with bone marrow depression, not caused by chloramphenicol itself. Apart from the question if this risk is estimated well [38], it can be avoided completely if the patient covers his skin if a dermal preparation has been applied and only use chloramphenicol as eye ointment 1 % at night. See also Sect. 10.​5.

If a light sensitive substance is administered via an infusion administration system, this should be covered, or made from opaque or completely light-resistant material.


22.2.5 Degradation of the Protein Structure


Many of the newly licensed medicines are biopharmaceuticals, which either are proteins or contain protein groups. Such pharmaceutical proteins are expected to become more and more available in the near future.

Examples of pharmaceutical proteins are: insulin, glucagon, somatostatin and analogues, erythropoeietin and analogues, interferons, interleukins, monoclonal antibodies, gonadorelin and analogues, colony stimulating factors, antithrombotic medicines, vaccines.

Proteins can be described by a primary, secondary, tertiary and quaternary structure, all of which can be affected by degradation processes. See also Sect. 18.​4.​1.

The primary structure of a protein consists of the arrangement into chains of amino acids. The number of amino acids per chain can vary from a few dozens to many hundreds.

The secondary structure is created by hydrogen bonds between the carbonyl group of one amino acid with the amine group of another. This structure manifests itself as an alpha helix or in a beta sheet. Within one protein, both alpha helixes and beta sheets can occur.

The tertiary structure is formed by the folding of peptide chains. Covalent and non-covalent bonds result in the positioning of the secondary structures in relation to each other. Covalent bonds are disulfide bonds between two cysteine units in the protein. Examples of non-covalent bonds are hydrogen bonds, ionic bonds and hydrophobic bonds in the protein. Which type of bond is formed, depends on the length of the peptide chain, pH, electric charge, polarity, and lipophilicity of (secondary) structural elements of the protein.

A quaternary structure is the arrangement of two or more protein subunits into a complex. These subunits may be identical but may also differ from each other. Interactions between subunits determine the three-dimensional orientation towards each other and therewith the structure of the whole protein (dimer, trimer, tetramer). The three-dimensional structure of proteins is essential for their biological efficacy and is highly sensitive to degradation.

The chemical bonds in proteins can be disrupted by:



  • Hydrolysis of amide bonds (proteolysis)


  • Deamination (cleavage of an unbound amino group of an amino acid)


  • Disulfide exchange (breaking of disulfide bonds and formation of new disulfide bonds with other cysteine units)

These degradation reactions are influenced by pH, temperature, and the presence of light and oxygen.

The spatial structure of proteins (tertiary and quaternary) can be disrupted by:



  • Aggregation (coagulation of proteins)


  • Denaturation (unfolding of proteins)


  • Precipitation


  • Adsorption (binding of lipophilic parts of the protein to surfaces)

Many factors influence the disruption of the spatial structure. Changes in temperature and pH can induce aggregation. The pH and ionic strength influence the charge of the amino acids and therewith the bonds and tertiary structure. In general, the higher the ionic strength, the greater the influence is on the structure. However, the effect of specific ions on the structure may vary.

In addition, the concentration of the pharmaceutical protein and the processes that they are subjected to, such as dissolving, suspending, and diluting, can be of influence. Shaking a vial can cause foam formation and denaturation. Shearing stress, as with the use of peristaltic pumps, can cause aggregation. Certain packaging materials are more absorbent than others.

Degradation of proteins can be prevented by not changing the composition of the solution of the protein, by not repackaging, by not shaking but gentle swirling of the solution, by not using peristaltic pumps, by avoiding large temperature differences, and by not heating the solution. Processes in the pharmacy in which proteins are involved are for example dissolving a freeze-dried pharmaceutical protein before use, or the preparation with a protein raw material like polymyxin.

Stability studies have demonstrated that monoclonal antibodies are chemically stable in daily practice. Rituximab and trastuzumab infusion are stable for 6 months when stored between 2 °C and 8 °C [39, 40]. Bevacizumab repackaged in syringes is stable for at least 3 months [41].



22.3 Microbiological Stability


Medicines must have a low micro-organism content and for some administration routes, the products have to be free of micro-organisms (sterile). The requirements are described in Sect. 19.​6.​2. The microbiological quality of a preparation can worsen after preparation despite a good starting quality. This happens when micro-organisms that are present grow (see Sect. 19.​2) or when micro-organisms from the environment contaminate the preparation during storage in the pharmacy or at home, and during use by the patient. The microbiological shelf life of a medicine depends on:



  • The initial degree of contamination


  • The probability of contamination from the environment


  • The suitability of the preparation for growth of micro-organisms (see Sect. 19.​4)

A good microbiological quality can be achieved by using starting materials of the right quality, by working hygienically, by using germicidal treatments, and by using clean or sterile packaging material (see Sect. 19.​5.​3).

The construction and volume of the stock packaging, the patient packaging, and the hygiene during repackaging from stock packaging to patient packaging determine the chance and amount of contamination. The formulation of the medicine and the storage conditions (temperature, humidity) determine the suitability for growth of micro-organisms. This is called the microbiological vulnerability (see also Sect. 22.3.3).

The factors packaging, hygienic handling, microbiological vulnerability, and storage conditions are discussed separately in the next paragraphs, but for the determination of a safe shelf life concerning the microbiological stability the interdependence of these factors should be considered. When for example a medicine is microbiologically vulnerable, the requirements for the construction of the container and the storage conditions must be stricter.


22.3.1 Packaging Material


For the storage of medicines in the pharmacy, storage in the primary container (as dispensed to the patient) is preferred over packaging in stock containers. The reason for this is obvious, since a primary container will not be opened before dispensing. Stock containers are frequently opened for repackaging the contents, and thus subject to contamination by the hands of the operator, utensils, and possibly by airborne micro-organisms.

When the use of stock containers is inevitable, the volume should be restricted to limit the number of times that the stock container will be opened. As a general rule, a stock container should contain no more than ten times the amount that is dispensed to an individual patient.

A multidose container should also ensure a minimum chance of contamination. This is especially important when the preparation is microbiologically vulnerable, or has stricter microbiological requirements (eye drops, creams) than preparations that are less vulnerable, such as water-free dermatological preparations.


22.3.2 Hygienic Handling


Hygienic handling means that contact of the medicine with sources of microbiological contamination are minimised.

Relevant sources of micro-organisms for pharmacy preparations are the human body, dirty utensils, and moist surfaces. These are more likely sources of contamination for medicines than airborne contamination.

The operator’s hands are an important source of microbiological contamination, as such, but also as a medium for transferring contamination coming from other body parts. This can be limited by wearing gloves during preparation. However, a study into the contamination of hard gelatine capsules via handling with bare hands showed that the contamination may be minimal in practice [42]. Despite these findings, the use of gloves becomes more and more common practice, not only from a microbiological perspective but also from an occupational health and safety perspective (see Sect. 26.​4.​3).

Hygienic handling is the most important factor for good aseptic handling, both in the pharmacy and during reconstitution on hospital wards (see Sect. 31.​3.​3).

Proper hygienic handling by the patient can be improved with a well-constructed container, such as a tube for a cream, a Gemo® dropper bottle for eye drops, or a pump sprayer for nose drops that uses air from the surroundings. In addition, the patient should be informed clearly and sufficiently about the importance of hygienic handling of the medicine (see Sect. 37.​7.​4).


22.3.3 Microbiological Vulnerability


A preparation is microbiologically vulnerable when it facilitates microbiological growth. Microbiological vulnerability can be investigated with a challenge test, a test for the effectiveness of the preservative. In this test, the degree of growth or eradication of micro-organisms in a formulation is investigated. The predominant reason to perform challenge tests is to test the effect of varying the formulation on the microbiological vulnerability. However, firm statements on microbiological vulnerability are difficult to make. Only a limited number of strains are tested and the question arises whether a single inoculation with a large number of micro-organisms is representative for a real contamination. Still, the Ph. Eur. contains requirements [43] for the minimal and recommended eradication rate, depending on the dosage form.

It is advised to test the microbiological vulnerability without the addition of a preservative. That way, it may be proven that the addition of a preservative is not always necessary. An example is a diacetylmorphine injection for multiple uses for heroin addicts. A solution of 150 mg/mL diacetylmorphine without preservative complied with the Ph. Eur. test [44].

Especially from research with food microbiology, knowledge is available of the factors that influence the microbiological vulnerability of medicines. The most important factors are the presence or absence of water, the presence or absence of antimicrobial agents, pH, environmental temperature and relative humidity. These factors are discussed separately.


22.3.3.1 Water


The presence of water is a prerequisite for the growth of micro-organisms (see Sect. 19.​2). This implies that in dry pharmaceutical preparations no growth of micro-organisms is to be expected, as spore forming micro-organisms reside in their spore form, whereas non-spore forming micro-organisms die. A trace of water, e.g. condensed water, may be sufficient for revival of spores. For this reason, a preservative agent is sometimes added to tablets that are used in tropical areas [45]. The chances of survival for various micro-organisms are dependent on the water content in the preparation. Some micro-organisms can only survive in a wet environment, whereas others are able to resist high salt concentrations or even grow in a (moist) powder. This knowledge is important for the investigation of the microbiological vulnerability of medicines. Preferably, those micro-organisms with the highest chance of survival in the product are used in studies.

A measure for the water content in a preparation is the water activity, as defined in the Ph. Eur. [46]. The water activity is the ratio between the water vapour pressure of a preparation at a certain temperature and the water vapour pressure of pure water at the same temperature. Therefore, water activity is the reciprocal of the osmotic value. Table 22.8 gives an overview of micro-organisms that can grow at a certain water activity, with pharmaceutical examples.


Table 22.8
Water activity, micro-organisms and pharmaceutical preparations (From [47] with permission)
























































Water activity

Micro-organisms that can grow at this water activity

Examples of pharmaceutical preparations with this water activity

1.00–0.95

Gram-negative rods, bacterial spores, some moulds

Eye drops, water containing creams

0.95–0.91

Most cocci, Lactobacillus species, vegetative forms of bacillaceae, some yeast
 

0.91–0.87

Most moulds

Simple Syrup

0.87–0.80

Most yeasts, Staphylococcus aureus

Sorbitol 70 % solution

0.80–0.75

Most halophile bacteria
 

0.75–0.65

Xerophile yeasts
 

0.65–0.60

Osmophile moulds

Starch with 18 % water

0.50
 
Extractum glycirrhizae crudum

0.40
 
Sodium lactate 60 % solution

0.20
 
Milk powder

0.0
 
Dried lactose

Micro-organisms cannot grow in a pharmaceutical preparation with a water activity below 0.5. Concentrated solutions, for example of saccharose, sorbitol, sodium chloride and urea, give micro-organisms little chance to grow, due to their low water activity. Simple syrup and sorbitol solution basically need no preservation, but nevertheless this is done for two other reasons. The first reason is that when simple syrup is used in oral mixtures, it is diluted. Preservation then gives an extra microbiological protection of the mixture. The second reason is that in the stock bottle of simple syrup condensed water can be formed at the surface and the vacant sides of the bottle, in which micro-organisms can grow.

Besides the water activity, the size of the water droplets in the water phase of an ointment can be important. Butter, which is not preserved, contains small water drops in which micro-organisms hardly grow, perhaps because the amount of food for the micro-organisms is limited. However, this theory does not apply to pharmaceutical preparations as well. In w/o ointment, growth of micro-organisms can occur when contaminated water is used (see Sect. 12.​5.​3).


22.3.3.2 Substances with an Antimicrobial Effect


Some active substances or excipients have an antimicrobial effect not only through reducing the water activity, but also through a specific antimicrobial mechanism. The best-known example is propylene glycol, but also ethanol, local anaesthetics, chlorpromazine hydrochloride, promethazine hydrochloride and essential oils exhibit a specific antimicrobial effect. In addition, the combination of weak antimicrobial effects of disodium edetate, borax, and boric acid appeared to justify prolongation of the shelf life of non-preserved eye drops over the standard limit of 24 h [48].


22.3.3.3 pH


Micro-organisms exist in numerous forms and shapes, so for every (extreme) pH value, types can be found that are able to survive and replicate under those specific conditions. However, micro-organisms that are commonly encountered during the preparation and use of pharmaceutical preparations do not grow outside the pH range of 3–9. There are a few examples of preparations with a pH outside this range, for example a ferrous chloride oral solution (pH 1.5) (Table 22.9) and a theophylline oral solution 30 mg/mL (pH 9.5) (Table 22.10). Such preparations thus require no preservation.


Table 22.9
Ferrous Chloride Oral solution 45 mg/mL [49]

















Ferrous chloride tetrahydrate (local standard)

 7 g

Citric acid monohydrate

 0.42 g

Sorbitol, liquid (crystallising)

 10.6 g

Syrup BP

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Jan 10, 2017 | Posted by in PHARMACY | Comments Off on Stability
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