Bacteria-proof

Filtration
 Sterilisation by Filtration
• Suitable for thermolabile medicaments.
• Limitation – medicament must be stable in solution.
• Aseptic process involves four stages :
a) Filtration of the solution through a bacteria proof filter.
b) Aseptic distribution of the filtered solution into previously
sterilized containers.
c) Aseptic closure of the container.
d) Testing of samples for sterility.
• Sterility testing are obligatory for injections sterilized by
filtration- bulk solution and final containers.

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Bacteria-proof Filtration Media
• There are four classes –
1) Sintered ceramics – made from finely ground porcelain or
from diatomaceous earth.
2) Fibrous pads – containing asbestos and wood cellulose.
3) Sintered glass – made from borosilicate glass.
4) Microporous plastic – prepared from cellulose esters,
particularly the acetate and nitrate.

• All types available in a range of porosities with the finest

suitable for removing bacteria (exception – all grades of
microporous plastics are bacteria proof)
3
Contd…
• Filtration through bacteria-proof filter is slow or negligible
under atm. pressure and is accelerated by applying pressure to
the liquid or vacuum to the clean side of filter.
• Features of Satisfactory Media :
 Sufficient porosity to allow adequate filtration rate, without
constant blockage.
 Yield nothing to the solution and must remove nothing but
organisms and insoluble material.
 Properties must not alter during moist or dry heat sterilization.
 Easy to assemble, or form an integral part of a leak proof
filtration unit.
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Contd…
 If not disposable, must be easy to clean by a method that does not
alter its porosity.
 If requires fitting into a metal holder (applies to disposable
media) the metal must not affect the solution being filtered.
• Efficiency of the filter medium must always be confirmed before
its initial use and at its regular intervals during its lifetime.

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A. Sintered Ceramics
• Sintered media -made by heating tightly packed particles of the
material in a suitable mould at a temperature below their m.p.
• Particles weld together at the points of contact, with the inter –
particle spaces intact. Porosity depends upon size of the particles.
• Sintered ceramic filters are in the form of hollow cylinders, usually
closed at one end and called candles.
• Candles present a large surface for filteration but retain an equally
greater volume inside the matrix at the end of the process.
• Max. pore diameter does not exceed 2.5 µm; can retain bacteria that
are not smaller than 1/3 of max. pore size.

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Contd…
I) Unglazed Porcelain : early filters like Pasteur-Chamberland -sinter a
mixture of kaolin (China clay) and quartz sand.
 Modern forms - finely ground unglazed porcelain, e.g. ‘Selas’ and
Doulton ‘Pasteur’ filters.
 Doulton candles range from 55 mm long by 10 mm diameter to 205 mm
long and 25 mm diameter. Walls 3 mm thick.

Characteristics–
 A candle 75 mm long by 15 mm dia with maximum pore dia of 2 µm
with pressure difference of 0.33 bar between the inner and outer faces,
will pass 550 ml of water/h until it begins to clog.
 An increase of 1 mm in wall thickness decreases flow rate by ~20%.
 Flow rate through a filter of maximum pore size 0.75 µm is only about
12% of the rate when the maximum pore is 2 µm.

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Contd…
Advantages
 Robust, stand up well to handling, to filtration pressures and
vacuum and to drastic cleaning methods.
 Only small amount of medicament adsorbed from solution
undergoing filtration.

Disadvantages :
 Less easy than most other types to fit into filtration units.
 Blocked quickly with microorganisms and foreign particles.
 Cleaning can be complicated.
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Contd…
Cleaning
1) New filters –
These are assembled in a filtration unit and
 Washed free from the dust of manufacture by drawing through a
large volume of water.
 Treated with hot dilute HCL acid to remove adsorbed impurities.
 Freed from acid by washing with tap and then apyrogenic water.
 Dried in a drying oven. If filters are stored wet pyrogens may be
produced within the pores.
2) Used filters –
 Immediately soaked in distilled water. They must not be allowed
to dry because the resulting deposits are very hard to dislodge.
 Scrubbed inside and out with a stiff brush.
 Flushed with apyrogenic water in the opposite direction.
 Dried .
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Contd…
• More drastic treatments necessary when reduced flow due to
blockage of the pores.
1) Chemical : Deposits are removed with oxidizing agents such as
hypochlorite, acid permanganate (0.5%) followed by sodium
thiosulphate (5%) or strong acid (nitric).
Chromic acid unsuitable because chromic ions are strongly adsorbed by the
filter and may not be completely removed.

2) Heat : Filter is dried thoroughly and put into a cold muffle-

furnace. Temp. raised slowly to 6000C and maintained for several
hours. Candle allowed to cool in the furnace, flushed to remove
residues, and finally dried.
• Each procedure can affect the porosity of the medium.
Contd…
• Cleaning of unglazed porcelain filters used in the manufacture of immunological products.
Contain high concentrations of sticky heat-coagulable protein and, therefore, cleaning can be particularly difficult.
Incubate the filter overnight at 370C in alkaline trypsin solution – the enzyme digests the proteins to simple water soluble degradation products that can be flushed away.

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Contd…
II) Kieselguhr: Contain high proportion of kieselguhr, a purified
siliceous earth consisting mainly of diatoms, hence the fineness of the
pores. Appropriate amount of asbestos and calcium sulphate media
added to produce different pore sizes.
•Commercial types - German Berkfeld filter and American Mandler.
•Thicker than unglazed porcelain and normally metal mounts attached
by cement.
Disadvantages –
Less robust than unglazed porcelain filters.
Should be handled gently, cleaned with soft brush, protected from
sharp pressure changes and allowed to cool completely upon
autoclaving.
Often, appreciable adsorption of the medicaments occurs.
After use for filtering oils they are particularly susceptible to
breakage and are very difficult to clean.
Contd…
Cleaning :
Choice is limited. Hypochlorites or permanganate can be used
but strong acids and heat treatment should be avoided.

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B) Fibrous Pads
• Soft pads 3 mm thick, usually round but occasionally square,
consisting largely of compressed asbestos.
• Used alone, asbestos becomes tightly compacted and the
filtration rate falls considerably. Therefore, it is usually
blended with wood cellulose which keeps the porosity high.
• Invented in Germany and marketed under trade name Seitz.
• Bacteria-proof grade is coded ‘EK’.
• Type ‘EKS’ is for sterilisation of aqueous injections.
• Type ‘EKS 1’ for colloidal, viscous and oily solution.
• Type ‘EKS II’ for serum.

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Contd…
• An equivalent made in England– the Ford Sterimat, the ‘SB’
quality of which is bacteria-proof.
• Sterimats are discs 3.6, 6 and 14 cm in diameter or in large
square sheets.

• Medium is spongy and fragile when wet and during filtration

must be supported on a perforated metal or glass disc in a
suitable holder.

• 6 cm disc filters about 200 ml of water/h when the pressure

difference between the faces is 0.33 bar.
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B. Fibrous Pads (contd…)
Advantages
 New pad is used each time. No risk of contaminating the
filtrate with either incompletely removed residue from a
previous filtration or traces of cleaning agents.
 Inexpensive. The holders are easy to dismantle and clean.
 Clog less easily than other media.
 More suitable than ceramics or glass for viscous solution.
 They can be used in the filtration method of sterility testing.

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Contd…

Disadvantages:
 Loose fibers may separate and contaminate the filtrate. Can
be remedied by attaching a tiny grade 3 or 4 sintered glass
clarifying filter to the stem of the holder (Seitz pads have an
impregnated coat on one surface which minimize separation
of fibers).
 Significant adsorption of medicament often occurs and can
cause serious loss of strength in solution of small volume.
Particularly marked with basic dyes and enzymes. With large
volume adsorption will take place from the first part of the
filtrate, so can be discarded.
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Contd…
 Sharp pressure changes may break the wet pads and cause
contamination of the filtrate with unfiltered solution. There
should be a buffer flask between the pump and the filter so that
pressure is not applied directly to the pad.

 Unsuitable for strongly alcoholic solutions (cause less swelling

and, therefore, organisms may pass through).

 Leach out alkalis which may precipitate alkaloids from

solutions of their salts and affect the stability of alkali sensitive
medicaments (insulin, adrenaline, apomorphine, thiamine).
Highly buffered solutions are unaffected (culture media).

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c) Sintered (Fritted) Glass
• Useful for sterilising small volumes.
• Manufactured by powdering high grade borosilicate glass in a ball
mill, separating the finest particles by air elutriation and sintering
appropriate sizes into discs in suitable moulds.
• Discs are fitted into Buchner-type or pipeline glass holders.
• Grade 5 (max. pore size not more than 2µm) is used for removing
bacteria.
• Filtration is slow through filters in which the max. pore is less than
1.5 µm.
• Thin discs can not withstand the pressure and vacua of bacterial
filtration, so it is customary to support thin layers of grade 5 on a
thicker layer of grade 3 (a clarifying grade) known as 5 on 3.
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Contd…
Advantages :
 If properly cleaned they yield nothing to the filtrate.

 Very little adsorption of medicaments.

 Volume of filtrate retained in the medium is significantly

less than with ceramic types.

 If desired, the holders can be fused into glass filtration units,

thus reducing the number of possible leakage points and
facilitating cleaning.
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Contd…
Disadvantages :
 The medium is unsuitable for large volume filtrations because
large discs are mechanically weak.
 The filters and, particularly, their holders are easily broken and
replacement is costly.
Cleaning : Comparatively easy. After use the filters are immediately
soaked in water and then the following are drawn through.
1. Water, to remove as much medicament as possible.
2. Hot (800C) concentrated sulphuric acid containing 1 to 2 % of
sodium nitrate, to oxidise deposits.
3. Water, until the medium is free from acid. Final flushing with
apyrogenic water.
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D. Microporous Plastics
• Thin membranes very tough when wet, with pores small enough to sieve
out bacteria from liquids or air.
• Available in variety of materials including regenerated cellulose (rayon),
polyvinyl chloride, nylon and apolyvinyl-acrylonitrile copolymer.
• Standard : MF-Millipore (a mixture of cellulose esters)
Sartorious Regular (cellulose nitrate) 0.22 µm
Gelman Triacetate Metricel (cellulose triacetate) 0.2 µm
• Available in 12 grades covering pore size from 8 µm to 10 nm.
• GS grade has a pore size of 0.22±0.02 µm, is generally used for bacterial
filtration (used for blood products).
• HA grade 0.45±0.02 µm, is used if very small bacterial contaminants
(pseudamonas) are absent.
Contd…
• All of these cellulose filters can be stored dry.
• Should be handled carefully (with flat tipped forceps) because
rather brittle.
• Become tough when wet.
• Can be sterilized by autoclaving for 15 min at 121 0C.
• Preferably, each membrane should be sterilized in its holder but,
if groups are sterilized they should be supported by interleaves
of thick absorbent filter pads, wrapped in paper and placed in a
flat tin (perforated).
• Certain grades of Millipore membranes are packed in an inner
envelope of polyethene and an outer of a polyester film and
sterilized by high energy electrons.
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Contd…
Advantages :
 Bacteria are removed mechanically by sieving, because pores
are so fine. Therefore there is little danger of organisms
gradually passing through the pores to contaminate the filtrate.
 Because the membranes are paper thin, adsorption of
medicaments is usually insignificant.
 A new disc is used for each filtration.
 The flow rate is high (most of the membrane consist of pores),
far less inter-pore matrix than in fibrous or sintered filters.
 MF-Millipore type HA passes 65 ml of water through each cm 3
per min at 200C ( 0.9 bars), GS grade passes 1/3 of this.

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Contd…

 Much greater differential pressure can be used. In others

differential is rarely allowed to exceed 1.5bars (7 bars).
 Do not yield particles or chemical substances to the filtrate.
 Organisms collected from gases or liquids can be counted and
studied by aseptically transferring the membrane to a thick pad
of absorbent filter paper (Whatman 17) previously saturated with
nutrient medium and incubating in a round tin or plastic box.

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Contd…
Disadvantages : compared with other media.
 Membrane clog easily because dirt and organisms are almost
entirely retained on the upper surface. Use of glass-fiber
prefilters can reduce this problem.
 Their chemical resistance is less. They are soluble in certain
organic solvents, e.g. ketones and esters ( advantage -
membrane can be dissolved away when separation of the
retained organism is desired)
 They are fragile. This problem is being tackled in various
ways, e.g. by presenting the filter in cartridge form and by
developing stronger membranes.

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Applications of the Various
Media
 Sintered filters for small volume injections because – are
easily assembled or fused into units, yield virtually nothing
to the filtrate and are not difficult to clean.
 Asbestos pads, preferred in industries (for injections
prepared from animal products) because – suitability for
large volumes, particularly when assembled in filter presses.
 Ceramics for immunological products.
 But these days all of these filters have been replaced by
membrane filters.

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 Filtration of Oils
• Filtration is a practical alternative to dry heat sterilization of oils.
• Use of dry Sterimats, Grade SB (fibrous pads) is recommended.
• Millipore corporation recommended the GS membrane and suggest
that the oil should be prewarmed, to increase the flow rate, and
preclarified to minimize clogging.
• For waxes – waxes preheated at 80 0C are passed through a GS
cartridge the housing of which is heated to 100 0C before filtration.

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Filtration as an Alternative to
a Heating Process
Manufacturing houses sometimes prefer it because –
 It is quick.
 Deleterious effects of normal heat sterilization are avoided.
 Simplification of the formula may be possible if the hazard of
heat exposure is eliminated.
Risk of bacteriological breakdown is much greater.
• Maintenance of asepsis during process; and good process and
• performance of the sterility tests.
• Should be used only when there is no alternative.
• Certainly not to replace the more reliable heating methods.
The Testing of Filters
This involves tests –

1. To show that the medium retains organisms satisfactorily. For

non-disposable filters must be done before a new filter is used
and at intervals during its life.
Proof of efficiency is obtainable in two ways.
a. Directly, by attempting to filter a suspension of a very small
organism (Bacteriological technique).
b. Indirectly, by finding the maximum pore size (Bubble pressure
technique).
2. To show that the flow rate of aqueous fluids is reasonably fast.
A filter with a satisfactory max. pore size might have high % of
much smaller pores and filter rate maybe extremely slow.

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1a. Bacteriological
Techniques
 Most convincing efficiency tests.
 A bacterial suspension of 24 to 48h broth culture of a
chromogenic strain, Serratia marcescens diluted to 25 times its
volume with nutrient broth.
 Suspension filtered at a pressure differential of nlt 0.5 bar.
 First 50 ml collected aseptically.
 Incubated aseptically at 250C for 5 days.
 There must be no growth of Serratia marcescens or any other
organism.

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Contd…
 S. marcescens chosen because it is small (0.3-1.3 μm long and
0.3-0.4 μm wide); aerobic; grows vigorously and, produces a
bright red pigment on incubation aiding detection.
 The first 50 ml is preferred because the medium will gradually
block as filtration progresses and the pores that at the beginning
will pass bacteria may not do so later.

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Contd…
Improvement of method (Sykes) :

1.Reduction of incubation time of initial culture to 24 h – Aged strains

are larger and, therefore, less able to penetrate a filter.
2.Increased dilution of the culture (200 times) and collection of smaller
volume (15 ml) - because prescribed suspension is rather concentrated
and quickly clogs filters of small surface area.
3. Increased pressure differential (0.9 bars) –modern pumps if not very
carefully used may subject filters to pressure as great as this.
4. Rejection of the filter only if Serratia marascens appears in the
filtrate – because other organisms have almost certainly come from a
leaky unit or bad technique and not from a faulty filter.
5. A test to confirm that the filter is not yielding inhibitory substances
Filtrates that are sterile after incubation should be lightly inoculated
with the test organism and re-incubated for 24 h.
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1b. Bubble-Pressure
Techniques
 Max. pore dia. can not be measured directly because of
irregularities within the pores but calculated from the pressure
necessary to force the first bubble of air through the medium
when it is wetted with a liquid of known surface tension,
because the first bubble forms at the pore of greatest diameter.
 Procedure : The filter is cleaned, dried, sterilised and soaked in
the test liquid.
• Water is used for clarifying filters but very high pressures are
required to blow air through bacteria proof types against water,
therefore, a liquid of lower surface tension is more satisfactory.
CCl4 is suitable (surface tension – 27 mN/m)

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Contd…
• Soaking expels the air from the pores. done by holding in
vacuum, admitting the liquid until the filter is covered.

• Then filter is connected to the compressed air system controlled

accurately e.g. an oil-sealed pump linked through shut-off and
needle valves.

• Candles are immersed in the shallow trough of the test liquid but
for disc filters it is usually more convenient to pour a thin layer
on the upper surface.

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 Bubble-Pressure Technique

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Contd…
• Pressure is applied very gradually via the needle valve.
• Pressure at which first bubble appears is noted (If the filter is
cracked or badly sealed to its holder large bubbles appear at
low pressure).
• Maximum pore size is calculated as–
D = (30 × 133.322) γ
ρ
where D = the pore size in micrometers
ρ = the pressure in N/m2
γ = the surface tension of the liquid in mN/m

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Contd…
Uniformity of Pore Diameter
 This determination shows that most of the pores are not
very much smaller than the max. and that the pores
distribution is even over the whole surface of the pad.

 After the first bubble breaks, in the determination of the

max. pore size, pressure is increased by a specified amount
e.g. by 0.2 bar for a grade 5 filter with CCl 4 and the bubbles
should escape evenly from all parts of the filter surface.

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Advantages of Bacterial
Filtration
 Preparation not heated – Method suitable for thermolabile
medicaments such as immunological preparations, blood products,
certain animal products (e.g. insulin) and enzymes ( e.g.
hyaluronidase).
 Both dead and living organisms are removed. In heating methods
the killed organisms remain in the preparation.
 The clarification and filtration can be carried out at the same time.
 Excellent method for the rapid supply of a small volume of a
parenteral solution in an emergency.
 Eye drops are usually heat sterilised but it is difficult to obtain a
dropper bottle that, when filled and closed, will withstand the
sterilization condition. Filtration (through membrane filter)
overcome this because the container is sterilized separately and in
two parts (bottle and dropper top).
Disadvantages of Bacterial
Filtration
 Aseptic technique is involved and necessitates – highly trained
staff and sterile equipment and facilities for preparation.
 Cannot be the last stage of the preparation because is only
applicable to bulk liquids.
 During the distribution of the filtrate into, and the closure of,
the final container there is a risk of accidental contamination.
Therefore, a bactericidal agent is advisable in all filtered
injections of small volume, both single and multi dose.
 Sterility tests, with their associated problems of design and
interpretation, are required on samples of every batch – both
bulk and unit dose.

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Contd…
 Although the no. of viruses present is reduced by adsorption,
viral sterility cannot be guaranteed.
 Faults in the filter media not immediately detectable. Non-
disposable filters must be regularly checked by bacteriological
efficiency tests or by pore-size determination.
 Units may leak if carelessly assembled.
 Adsorption of medicaments may occur. Most significant with
kieselguhr candles and to a lesser extent, fibrous pads.
 If filtration is prolonged, organisms may pass through the
media that do not retain by sieving e.g. with sintered-glass
filters contamination of nutrient solution occurs after 18 h.
Contd…
 Most of the media have disadvantages :
i. Unglazed porcelain – cleaning, retention of a considerable
volume of the filtrate in the medium,
adsorption.
ii. Kieselguhr – adsorption.
iii. Fibrous pads – yield particles and alkali, adsorption.
iv. Glass – weak when large size.
v. Membranes – clogging.
 Filtration is unsuitable for suspension.
 Only be used if the medicament is stable in the solvent at normal
storage temperature.
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Mechanism of Bacterial
Filtration
A. Mechanical Sieving – fine porosity filters, such as
microporous plastics, retain organisms because their pores
are smaller than bacteria. If the sieving process was the
only mechanism, sintered media and fibrous pads, in which
the majority of pores are larger than bacteria, would not be
bacteria proof.

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Contd…
B. Retention on Pore Irregularities : an organism passing along a pore
in a sintered or fibrous filter travels a tortuous path that has a very
uneven surface. There are about 200 irregularities/cm of pore in an
unglazed porcelain candle. Bacteria may be stopped by or trapped
in these hazards. This suggests for the penetration of sintered and
fibrous media after prolonged filtration. Bacteria, originally
arrested on the projections in the upper part of the filter become
dislodged as filtration proceeds and, after a series of further
temporary retentions, eventually appears in the filtrate.

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Contd…
C. Imbibition of Water : cellulosic fibers in fibrous pads
imbibe water from aqueous solutions. This causes the pad
matrix to swell, with consequent reduction in the size of the
interstices and more efficient retention of bacteria. Strongly
alcoholic solutions cause less swelling and, therefore,
organisms may pass through.

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Contd…
D. Electrostatic Attraction : Retention of particles by
electrostatic attraction is well-known for certain plastics
(e.g. polythene) and synthetic fibers. The same force may
assist the retention of bacteria with in a filter.
• This theory is difficult to substantiate, it would hold if
bacteria were positively charged, because ceramic, glass and
asbestos fibers carry negative charge but acidic groups
predominate in all bacterial species.
• Overall charge on the organism may not be an important
factor, and retention may take place through the basic groups
of certain cell components, particularly proteins.

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Contd…
• The pH of the filtering solution would be expected to
influence this mechanism by altering the basicity or acidity
of the proteins.

• Sterimats SB can be made permeable to bacteria by soaking

in oil before filtering an aqueous suspension of organism.
Oil insulates the charge on the filter.

• Surface active agents, e.g. certain soaps and bile salts,

increase the permeability of the filters, possibly by affecting
the surface charge.

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Electrostatic Attraction

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