Aseptic Processing

Aseptic processing is concerned with the preparation of those sterile products that cannot be
subjected to a terminal heating process because the medicaments they contain are thermolabile.

Sterilization by filtration:
Filtration through a bacteria-proof filter is a suitable method for the sterilization of injections
containing thermolabile medicaments. However, there is an important limitation – the medicament
must be stable in solution.
The process involves four stages –
1. Filtration of the solution through a bacteria-proof filter.
2. Aseptic distribution of the filtered solution into previously sterilized containers.
3. Aseptic closure of the containers.
4. Testing of samples for sterility.

Bacteria-proof Filtration Media:

The features of a satisfactory medium are:
1. The porosity of the material must be sufficient to allow an adequate filtration rate for the job
in hand, without constant blockage.
2. It must yield nothing to the solution and must remove nothing but organisms and insoluble
material.
3. Its properties must not alter during moist or dry heat sterilization.
4. It must be easily assembled into, or form an integral part of, a leak-proof filtration unit.
5. If not disposable, it must be easy to clean by a method that does not alter its porosity.
6. If it requires fitting into a metal holder, the metal must not affect the solution being filtered.

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. Micro-porous plastics – prepared from cellulose esters, particularly the acetate and nitrate.

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1. Sintered ceramics:
Sintered media are made by heating tightly packed particles of the material in a suitable mould at a
temperature below their melting point. The particles become welded together at the points of contact,
which increase in area but do not grow large enough to obliterate the inter-particle space. Therefore,
a porous mass result in which the porosity depends on the size of particles used.

Sintered ceramic filters are in the form of hollow cylinders, usually closed at one end and called
candles. The other media are used in the form of discs. Candles present a larger surface to the
filtering liquid but retain a greater volume inside the matrix at the end for the process.

Usually a sintered medium is bacteria-proof if the maximum pore diameter does not exceed 2.5μm.
However, the efficiency of this type of medium must always be confirmed before its initial use at
regular intervals during its lifetime.

a) Unglazed Porcelain:
Earlier filters of this type were produced by sintering a
mixture of kaolin and quartz sand, but modern forms
are made from finely ground unglazed porcelain.
Examples are the Doulton ‘Pasteur’ and ‘Selas’ filters.

Stock sizes of Doulton candles range from 55 mm long

by 10 mm diameter to 205 mm long and 25 mm
diameter. Their walls are about 3 mm thick. Some sizes have glazed porcelain mountings to
facilitate incorporation into filtration units. Unmounted candles with a glazed top are also
available. The maximum pore size is usually between 2 & 2.5 μm.

Advantages:
1. These filters are robust. They stand up well to handling, to filtration pressures and vacua and
to drastic cleaning methods.
2. Only small amounts of medicaments are adsorbed from solutions undergoing filtration.

Disadvantages:
1. They are less easy than most other types to fit into filtration units.
2. They become blocked rather quickly with microorganisms and foreign particles.

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 3. Cleaning can be complicated.

b) Kieselguhr:
These candles contain a high proportion of
kieselguhr, a purified siliceous earth consisting
mainly of diatoms, hence the fineness of the pores.
Media of different pore sizes are produced by
including appropriate amounts of other substances
such as asbestos and calcium sulphate.
The earliest and still the most important commercial type is the German Berkfeld filter. Another
is the American Mandler.
Kieselguhr candles are thicker than unglazed porcelain types and normally have glazed porcelain
or metal mounts attached by cement.

Disadvantages:
1. They are less robust than unglazed porcelain filters. They should be handled gently, cleaned
with a soft brush, protected from sharp pressure changes and allowed to cool completely
before removal from the autoclave.
2. Often, adsorption of medicaments occurs.
3. After use for filtering oils they are particularly susceptible to breakage and very difficult to
clean.

2. Fibrous pads:
These are soft pads about 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.
Fibrous pads are used once and discarded.
They were invented in Germany and marketed under the trade name of Seitz. The bacteria-proof
grade is coded ‘EK’.

Advantages:
1. A new pad is used each time, so there is no risk of contamination. The pads are inexpensive and
the holders are easy to dismantle and clean.
2. They clog less easily than other media.

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3. They are more suitable than ceramic of glass for viscous solutions.
4. They can be used in the filtration method of sterility testing.

Disadvantages:
1. Alkali may be given to the filtrate which may be sufficient to precipitate alkaloids from solutions
of their salts and affect the stability of alkali-sensitive medicament such as insulin, the posterior
pituitary hormones, adrenaline, apomorphine and thiamine.
2. Loose fibers may separate and contaminate the filtrate.
3. Significant adsorption of medicaments often occurs and can cause serious loss of strength in
solutions of small volume.
4. Sharp pressure changes may break the wet pads and cause contamination of the filtrate with
unfiltered, unsterile solution.
5. Unsuitable for strongly alcoholic solution.

3. Sintered (fritted) Glass:

This medium is useful for sterilizing small volumes. It is 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.

Advantages:
1. If properly cleaned they yield nothing to the filtrate.
2. There is very little adsorption of medicaments.
3. The volume of filtrate retained in the medium is less than with ceramic types.
4. 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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Disadvantages:
1. The medium is unsuitable for large-volume filtrations because large discs are mechanically
weak.
2. The filters and particularly, their holders are easily broken and replacement is costly.

4. Micro-porous Plastics:
Bacteria-proof filters of micro-porous plastics are thin membranes, very tough when wet, with pores
small enough to sieve out bacteria from liquids or air.
They are usually known as membrane filters, which are available in a variety of materials including
regenerated cellulose (rayon), polyvinyl chloride, nylon and a polyvinyl-acrylonitrile copolymer.
Millipore filters are good examples of membrane filters where a mixture of cellulose esters is used.
Millipore filters are available in 12 grades covering pore sizes from 8μm to 10nm. The GS grade,
which has a pore size of 0.22 ± 0.02 μm, is generally used for bacterial filtration, but an alternative
grade, HA, of pore size 0.45 ± 0.02 μm is satisfactory when very small bacterial contaminants are
absent.
All the cellulose filters can be stored dry. In this condition they should be handled carefully because
they are brittle. They can be sterilized by autoclaving for 15 min at 121ºC.

Advantages:
1. Bacteria are removed mechanically by sieving because the pores are very fine. Therefore, there is
little danger of organisms gradually passing through the pores to contaminate the filtrate. (This
may occur with ceramic, asbestos-cellulose & glass types if filtration is prolonged).
2. Adsorption of medicaments is usually insignificant as the membranes are paper thin.
3. A new disc is used for each filtration.
4. The flow rate is high because most of the membrane consists of pores; there is far less inter-pore
matrix than in fibrous or sintered filters. A type HA Millipore membrane passes 65 ml of water
through each cm2 per minute at 20 ˚C & 0.9 bar pressure. Whereas with fibrous pads the flow
rate is only 6% of HA rate.
5. Much greater pressure differentials (7 bars or more) can be used with these membrane filters.
That’s why these are 40 times faster than conventional filters of equivalent area & retention
efficiency.
6. They do not yield particles or chemical substances to the filtrate.
7. They can be used for sterility testing by filtration.
8. Organisms collected from liquids or gases can be counted and studied.

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Disadvantages:
Compared with the older types of media -
1. Membranes clog more easily because dirt & organisms are almost entirely retained on the upper
surface. Thin glass-fibre paper prefilters are available to reduce this problem.
2. Their chemical resistance is less. They are soluble in certain organic solvents e.g. ketones &
esters. But membranes of regenerated celluloses have high resistance to common organic
solvents.
3. They are fragile. This problem is being tackled in various ways, e.g.
a) By presenting the filter in cartridge form: The membrane is wrapped round a cylinder
of polypropylene or stainless steel and protected by a hard-porous sleeve.
b) By developing stronger membranes: Examples include: Acetonitrile-polyvinychloride
copolymer. This can be used in pleated form to present a very large filtration area.

The Testing of Filters:

This involves tests -
1. To show that the medium retains organisms satisfactorily:
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 and assuming that if this is no greater
than the maximum, in previously satisfactory filters of the same type filter; will be
bacteria-proof (Bubble pressure technique)
2. To show that the flow rate of aqueous fluids is reasonably fast:
A filter with a satisfactory maximum pore size might have a high percentage of much smaller
pores and therefore filter extremely slowly.

Bacteriological Technique:
A suitable bacterial suspension is made by diluting a suitable quantity of a 24 to 48-h broth culture
of a chromogenic (pigment-producing) strain of Serratia marcescens (formerly known as
Chromobacterium prodigiosum) to 25 times its volume with nutrient broth. This organism is chosen
because it is small (0.3 to 1.3 μm long and 0.3 to 0.4 μm wide) and aerobic; it grows vigorously and,
if incubated at 25ºC, produces a bright red pigment that aids its detection.

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The filter is assembled into a unit which is then sterilized. Afterwards the unit is set up aseptically
and the suspension is filtered at a pressure differential of not less than 0.5 bar.
50 ml are collected aseptically; preferably the first 50 ml because the medium will gradually block as
filtration progresses and pores that at the beginning will pass bacteria may not do so later.
The container is aseptically closed and incubated at 25ºC for 5 days. There must be no growth of
Serratia marcescens or any other organism.

Advantages of bacterial filtration:

1. Since the preparation is not heated, the method is suitable for thermolabile medicaments such as
immunological preparations, blood products, certain animal products (e.g. insulin) and enzymes
(e.g. hyaluronidase).
2. Both dead and living organisms are removed. In heating methods the killed organisms remain in
the preparation.
3. If the solution has been prepared carefully, to reduce foreign particles to a minimum, and the
volume is small, clarification and filtration can be carried out at the same time.
4. If sterile filters, units and containers are always kept available, filtration is an excellent method
for the rapid supply of a small volume of a parenteral solution in an emergency.
5. Eye drops are usually heat sterilized but it is difficult to obtain a dropper bottle that, when filled
and closed, will withstand the sterilization conditions. Filtration overcomes the problem because
the container is sterilized separately and in two parts (bottle and dropper top).

Disadvantages of Bacterial Filtration:

1. Aseptic technique is involved and requires-

a) Highly trained staff who are fully aware of the limitations
b) Sterile equipment and facilities for its preparation.
2. It cannot be the last stage of preparation because it is only applicable to bulk liquids. There is a
risk of accidental contamination during the distribution of the filtrate into the final containers.
Therefore, a bactericide is advisable in all filtered injections of small volume, both single and
multi-dose.
3. Sterility tests, with their associated problems of design and interpretation, are required on
samples from every batch. Except in an emergency, issue is not permitted until the tests have
been passed.

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4. Viral sterility cannot be guaranteed. However, because of the absence of living cells, essential
for their growth, viruses cannot multiply.
5. Faults in the media are not immediately detectable.
6. Units may leak if carelessly assembled. It is advantageous to have few joints and to use pressure
rather than vacuum.
7. Adsorption of medicaments may occur.
8. If filtration is prolonged, organisms may pass through the media that do not retain by sieving.
9. Most of the media have disadvantages, e.g.

Unglazed porcelain -Cleaning

-Retention of a considerable volume of the filtrate in the medium
-Adsorption
Kieselguhr -Adsorption
Fibrous pads -Yield particles and alkali
-Adsorption
Glass -Weak when large
Membranes -Clogging

10. Filtration is unsuitable for suspensions.

11. It can only be used if the medicament is stable in the solvent at normal storage temperature.

Mechanisms of Bacterial Filtration:

1. Mechanical Sieving:
Fine porosity filters, such as micro-porous plastics, retain organisms because their pores are
smaller than bacteria. If this 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.

2. 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. It has been estimated that there are about 2000 irregularities/cm of pore in
an unglazed porcelain candle. Bacteria may be stopped by or trapped in these hazards.

3. Electrostatic Attraction:
This theory offers an idea that the filters may retain bacteria by electrostatic attraction. But, this
theory is difficult to prove. Ceramic, glass and asbestos filters would hold if the bacteria were

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 positively charged, because they all carry negative charges but, in fact, negatively charged acidic
groups predominate in all bacterial species.

However, the overall charge on the organism may not be the important factor and retention may
take place through the basic groups of certain cell components, particularly proteins.
Some surface active agents, e.g. certain soaps and bile slats, increase the permeability of filters,
possibly by affecting the surface charge.

4. Imbibition of Water:
The 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 (intervening spaces) and
more efficient retention of bacteria. Strongly alcoholic solutions cause less swelling and,
therefore, organisms may pass through.

Glossary:

Sintering is the process of compacting and forming a solid mass of material by heat and/or pressure
without melting it to the point of liquefaction. The atoms in the materials diffuse across the
boundaries of the particles, fusing the particles together and creating one solid piece.

Diatomaceous earth, also known as diatomite, or kieselgur/kieselguhr, consists of fossilized

remains of diatoms, a type of hard-shelled algae which is easily crumbled into a fine white to off-
white powder. It has a particle size ranging from less than 3 micrometres to more than 1 millimetre,
but typically 10 to 200 micrometres. The typical chemical composition of oven-dried diatomaceous
earth is 80 to 90% silica, with 2 to 4% alumina (attributed mostly to clay minerals) and 0.5 to
2% iron oxide.

Asbestos is a set of six naturally occurring silicate minerals, which have common their crystal habit:
long, thin fibrous crystals, with each visible fiber composed of millions of microscopic "fibrils" that
can be released by abrasion and other processes. They are commonly known by their colors, as blue
asbestos, brown asbestos, white asbestos, and green asbestos.

Fritted glass is finely porous glass made by sintering together glass particles into a solid but porous
body. Applications in laboratory glassware include use in fritted glass filter items, scrubbers, or
spargers. Other laboratory applications of fritted glass include packing in chromatography
columns and resin beds for special chemical synthesis.

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Porcelain is a ceramic material made by heating materials, generally including kaolin, in a kiln to
temperatures between 1,200 and 1,400 °C (2,200 and 2,600 °F). The toughness, strength and
translucence of porcelain, relative to other types of pottery, arises mainly from vitrification and the
formation of the mineral mullite within the body at these high temperatures.

Regenerated cellulose is a class of materials manufactured by the conversion of natural cellulose to

a soluble cellulosic derivative and subsequent regeneration, typically forming either a fiber
(via polymer spinning) or a film (via polymer casting)

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