Contamination control and Preservatives

what is the purpose of preservatives?

protect against:

• residual contamination not excluded by GMP

• contamination introduced during use

what types of products are preservatives usually used in?

• formulations which aren’t ‘self’ preserved e.g. ones of a low pH, dry formulations

• preparations in multidose containers e.g. creams

• aqueous preparations

• non-terminally sterilised products

what considerations need to be taken into account when selecting a preservative?

• type of formulation and container

• ingredients → interactions, compatibility

• physicochemical properties

• route of administration e.g. cannot use preservatives in intra-spinal injections

why can’t you use large doses of preservatives?

ADRs can occur

describe the ideal activity of preservatives

• ideally want to use a broad spectrum preservative which is active again gram positive and negative bacteria, yeast fungi etc

• but many show limited activity :. combinations are used to try to achieve a broad spectrum cover

give an example of a broad spectrum preservative

bronopol

give an example of a preservative with limited spectrum activity

cetrimide

how does pH affect the activity of some preservatives?

• have to consider whether preservative is active in its unionised or ionised form

• then have to look at its pKa and establish whether it’s going to be unionised or ionised in the body

when considering benzoic and sorbic acids as preservatives, what is their pKa? when are they active? in which formulations are they used in?

• pKa = 4.2-4.5

• active in unionised form :. in lower pHs

• only used in formulations at pH 3-6

when considering phenolics as preservatives, what is their pKa? when are they active? in which formulations are they used in?

• pKa ~ 10

• active in unionised form

• effective at lower pHs → inactive above pH 9

why are phenolics, sorbic acids and benzoic acids active in their unionised form?

exert action by penetrating into microbe :. unionised means they are able to cross cell membrane better

when considering cationic (positively charged) preservatives, when are they active? in which formulations are they used in?

• active in their ionised positively charged form

• active in formulations above pH 4.5

give an example of a cationic preservative

chlorhexidine

why are cationic preservatives active in their ionised positively charged form?

interact with cell membranes → microbial membranes are often negatively charged :. needs to be ionised to carry positive charge and interact

what are the 2 ways in which a surface active agents in a formulation impact which preservative is selected?

• compatibility

• solubilisation

how do surface active agents in a formulation impact which preservative is selected in terms of compatibility?

• can have positively charged or negatively charged surface active agents

• :. needs to choose a preservative which is compatible with the surface active agents

• e.g. phenols, benzoic and sorbic acids would be neutralised by cationic surface active agents

• e.g. cationic preservatives would be neutralised by anionic (negatively charged) surface active agents

how do surface active agents in a formulation impact which preservative is selected in terms of solubilisation?

• above the critical micelle concentration you get a 2 phase solution → aqueous and micellar phases

• if the preservative is lipophilic, it will partition into the micellar phase

• means there is a lower concentration of the preservative in the aqueous phase

• if concentration of preservative drops below its Minimum Inhibitory Concentration (MIC) level then it is no longer working in the aqueous phase now

• important because microbes grow preferentially in the aqueous phase

what is the Minimum Inhibitory Concentration (MIC) level?

lowest concentration of the preservative required to inhibit the visible growth of a microorganism

what does this graph show?

• parabens are lipophilic

• as you increase the concentration of tween 80, the proportion of micelles increase :. more paraben preservative is partitioning into the micellar phase

• blue line = MIC level

• to maintain the MIC level during the increase in the concentration of tween 80, you’d need to add increasing amounts of parabens corresponding to the red line

• can only go so far up red line before dose related paraben toxicities show up

how do solids in the formulation (e.g. API, thickening agents, suspending agents) affect preservative choice?

preservative may adsorb onto the solid particle surface :. no longer available :. reduces aqueous concentration

• e.g. chlorhexidine adsorbs onto kaolin

what does the concentration coefficient show?

shows reduction in preservative activity upon dilution

why is partitioning behaviour of the preservative important?

in multiphase systems, the preservative is partitioned between phases :. an adequate concentration must be maintained in the aqueous phase

why is the solubility of a preservative important?

• preservatives need to be soluble in the aqueous phase of a formulation to ensure antimicrobial activity

• also needs to be lipophilic to enable partitioning through the microorganisms membranes

e.g. parabens increase in activity with an increase in the ester chain length (i.e. more lipophilic = more active) BUT solubility decreases :. use a combination of parabens at saturation concentration

what can be done to enhance solubility of lipophilic preservatives (e.g. parabens)?

use cosolvents

give examples of cosolvents

• ethanol

• propylene glycol

• glycerol

how does temperature affect preservative activity?

generally increases preservative activity → important when extrapolating activity data from room temperature to storage temperature

• but also increases bacterial growth

what is used to measure the affect of temperature on preservative activity?

Q10 → coefficient for the change in activity per 10ºC change in temperature

how does stability affect preservative choice?

product shelf life is ideally 2-3 years → formulation has to remain stable for the entire duration of this

• not all preservatives are suitable e.g. bronopol is unstable at alkaline pH → 50% decomposed in 2 months at pH 8, some are photolabile etc

what effect does interaction with the container have on preservative choice?

adsorption to the container means the concentration of preservative is reduced in aqueous phase :. needs to be compatible

which packaging material is fairly inert?

glass

what other considerations are ideal for a preservative?

• non-irritant (especially for topicals)

• non-sensitising

• non-toxic

• colourless

• odourless

• no taste

• inexpensive

how to you determine microbial quality assurance? what is this required for?

preservative efficacy test → required test for all vulnerable products

how does the preservative efficacy test work?

• take product in its final container and inoculate it with high quantities of S. aureus, P. aeruginosa, A. niger, C. albicans

• after period of time, samples of taken

• enumerate and identify the bugs (need to neutralise preservative first :. can filter :. bugs remain on filter or add something to inactivate the preservative)

• will show how effective preservative is in its final container

what types of organisms are S. aureus, P. aeruginosa, A. niger, C. albicans?

• S. aureus → gram positive bacteria

• P. aeruginosa → gram negative bacteria

• A. niger → mould (yeast)

• C. albicans → fungi (yeast)

what are the drawbacks of the preservative efficacy test (PET)?

• 4 organisms unlikely to truly represent all possible contminants

• only a single challenge which is unrealistic for multidose containers

• lab strains of bacteria will have different patterns of resistance of wild strains contaminating products during use or manufacture

• 28 d cannot be extrapolated to an expected shelf life of 2-3 y

• inoculum size is unrealistically high, but 10⁶ chosen to enable an end-points to be accurately determined i.e. 10³

• sample of 1 ml will be unable to detect <1 organism / ml but just one organism can replicate and cause spoilage