Aseptic Manufacturing

• Clean room, validation of clean room.

• HVAC system.
• Pyrogen and depyrogenation.
• Filling and sealing of sterile products.
• Manufacturing of SVP and LVP.
• Cleaning and sterilization of containers and closures.
• Clean in place (CIP) and sterilize in place (SIP) system.

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 Sterile Production
Sterile Products are dosage forms of therapeutic agents that are free of
viable micro organisms.
 Example: Parenterals, Ophthalmic Products & Irrigating Solutions.
 Sterile products are most frequently solutions or suspensions, but may
even be solid pellets for tissue implantation.
 The term “parenteral” means to deliver the medication via a route other
than through digestive tract. Two Greek wards, Para- Outside, enteron-
Intestine.
 The most common route used to deliver parenteral medication is
through injection.
 Various routes of Parenteral preparations – Intravenous, Intraspinal,
Intra muscular, subcutaneous , Intradermal, etc.
 Routes of administration
 Intravenous route (IV):
• Advantage: -rapid action ,-optimum blood levels, -life saving
• Disadvantage-it cannot be retrieved.
 Intramuscular route (IM) –less rapid but generally longer lasting than IV. -administered
intramuscularly. -performed deep into skeletal muscles
 Subcutaneous route (SC) – under the skin. - Small amounts medication. - Absorbed
more slowly than IV.
• Example -Some vaccines and allergy shots . Some pain medications like morphine and
hydromorphone .
 Intradermal route(ID) – A shallow or superficial injection. -injected into the corium
or dermis -located between epidermis & hypodermis.
• Example- Various agents for diagnostic detections, de-sensitisation or immunisation.
Tuberculose tests, Allergy tests
 Small & Large Volume Parenteral
Based on volume Sterile liquid products are classified as SVP’s & LVP’s.
Small Volume Parenteral (SVP) :
o The volume is generally less than or equal to 100ml.
o They are supplied in a single or multiple dose (Vial, ampoule).
o Example: pharmaceutical products (ophthalmic & parenterals), Diagnostic
agents, allergenic extracts ,radiopharmaceuticals & biologicals.
Large Volume Parenteral (LVP)
o The volume is generally more than 100ml or more (1 litre).
o Packaged in a single-dose container & deliver through IV route.
o Example: blood collecting units with anticoagulants, peritoneal.
dialysates, irrigating solutions, diagnostic agents & IV fluids (Saline
solutions (electrolytes), dextrose solution (nutrition), ringers solutions, ).
 Formulation of parenterals
1. Vehicle –
o The most frequently used vehicle is water, as it is the vehicle for all natural body
fluids.
o Tests for checking water quality - Total solid content, organic & inorganic substances ,
electrolytic measurement of conductivity. (Less than 1micro mho)
o The another vehicle is sterile WFI (sterilized by a thermal method).
o The cosolvents such as Aqueous vehicle – Ethyl alcohol, PEG, PG.
2. Non aqueous vehicles –
o must be non irritating, non toxic & must not exert an adverse effect on ingredient of
formulation.
o Checking of physical properties - density, viscosity, pH, miscibility, boiling point, low
vapour pressure & polarity etc.
o Example: fixed oils (corn oil, peanut oil, cotton seed oil)
 Formulation of parenterals
3) Solutes (API) –
o It mustd be free from pyrogenic & microbial contamination.
o Some solutes sterile grade is available e.g. – Vitamin-C, calcium gluconate.
o Special parenteral grades are available.

4) Added substances –
o It includes solubilisers, Osmatic pressure adjuster, antioxidants, chelating
agents, buffers, tonicityrs builders, antibacterial agents, hydrolysis inhibitors,
antifoaming agents.
 The preparation of a parenteral solution involves the dissolution of all the
ingredients into an appropriate solvent system.
 Water for Injection (WFI)
WFI –
• It is non-pyrogenic distilled water.
• Use in the preparations of medicines for parenteral administration.
• The USP defines this as highly purified waters containing less than 10
CFU/100 ml of Aerobic bacteria.
• WFI is obtained by distilling potable water or purified water from a
neutral glass or prepared by reverse osmosis.
Sterile WFI –
• It is WFI distributed in suitable containers of glass or any other material,
sealed & sterilized by heat under conditions that ensure that water
remains non-pyrogenic.
 Common excipients used in parenterals
Concentration range (%) Excipients Sl No
Anti microbial preservatives 1
0.5-10.00 Benzyl alcohol
0.001 Phenyl mercuric nitrate
0.001-0.02 Thimerosal
0.005-0.035 Propyl paraben
Solubilisers, wetting agents/emulsifiers 2
0.01 Dimethyl acetamide
14.6-25.0 Ethanol
0.61-49.0 Glycerol
0.01-50.0 PEG 300
Buffers 3
0.22 Acetic acid
0.5 Citric acid
1.6 Maleic acid
0.1 Lactic acid
 Common excipients used in parenterals
Concentration range (%) Excipients Sl No
Tonicity modifiers 4
0.14-5.0 Lactose
0.4-2.5 Mannitol
2.0 Sorbitol
Varies Nacl
Suspending Agent 5
2.0 Gelatin
0.2 Pectin
2.7-4.0 PEG-4000
Anti oxidant 6
0.02-0.1 Ascorbic Acid
0.005 Thiourea
0.005-0.002 BHT
 Common excipients used in parenterals
Concentration range (%) Excipients Sl No
Local Anesthetics 7
1.0 Procain Hcl
5.0 Benzyl Alcohol

Stabilizer 8
1.25-2.5 Niacin
0.4 Sodium Caprylate
1.5-2.4 Glycin
Chelates 9
0.00368-0.05 EDTA disodium
0.01 EDTA tetrasodium
 Manufacture of SVP’s
Equipment & facilities management –
• Washing & Sterilization equipment (glass, plastic & rubber components)
• Mixing equipment to manufacture the bulk product
• Filtering equipment to clarify & or sterilize product
• Storage tanks to hold the bulk product prior to sub division
• Filling or subdividing equipment
• Stoppering & Capping equipment
• Terminal sterilization equipment.
Preparation of facilities – a) cleaning of service areas b) preparation of clean room
areas c) preparation of sterile room d) preparation of equipment.
Preparation of packaging components – A sterile package consist of primary &
secondary packaging components. Examples of primary packaging components are
ampoules, vials, syringes, syringe cartridges, squeeze bottles & rubber/plastic stoppers.
A box or a shrink wrap is an secondary packaging components.
 Manufacture of SVP’s
Operation-I (Non sterile)

1. WFI - In a clean, vented, glass lined/stainless steel pressure tank. (excess of 10% of final
volume). Seal the pressure tank.
2. Sterilize - Heat at 121◦C for 20 min. - Cool to 60◦C .
3. Keep approx. 30% WFI, in a separate vented SS tank/vessel of suitable capacity for final
volume adjustment
4. To remaining WFI (at 60◦C ), from step-2, add & dissolve with stirring to monobasic &
dibasic sod. phosphate.(Bufering agent)
5. Cool to room temperature (25-30◦C ), then add and dissolve with stirring water soluble
drug & preservative, check the pH of solution if required adjust to with approx 1N
NaoH solution.
6. Adjust the final volume with WFI & mix well.
 Manufacture of SVP’s
Operation-II (Sterilization by filtration)
1.Sterilise the bulk solution, by filtration through a sterile sterilizing membrane, with
an appropriate non shedding preclarification filter.
2. Collect the sterile filtrate directly from sterilizing membrane into a sterile, clean,
closed, vented, SS tank vessel.
Operation-III (Sterile Filling or subdivision)
1. Aseptically subdivide sterile bulk solution into an appropriate sterile container.
2. Aseptically apply sterilized closure systems to container & seal.
3. Inprocess check: Sample across filtering operation at intervals determined by QC
standards for sterility tests & volume fill checks.
4. Visual Inspection –Check defects & particulate against a well lighted black & white
background.
5. Submit samples to QC for assay.
 Preparation of sterile powders
Preparation of sterile powders
o Injections are also packaged as dry solids (Vial) rather than in conjuction with solvent
because therapeutic agent is unstable in the presence of liquid component.
o These dry powders are packaged in the final container to be reconstituted, generally to
a solution or less frequently a suspension.
o The method of sterilisation of powder - dry heat or other appropriate method.
o Sometimes liquid is packaged with dry powder for use at the time of reconstitution.
o This liquid is sterile & may contain some of the desired additives such as buffer.

Containers for parenterals

1. Glass – It includes ampoules, vials, 1 litre bottle or more capacity
2. Plastic – 1 litre capacity or more
3. Rubber – Closure material sealed with aluminium
 Filling & Sealing of Parenterals
Filling equipment for liquids –
 A measured volume of liquid is forced through the orifice of a delivery tube designed
to enter the constricted opening of a container.
 The size of the delivery tube depends on the opening in the container, the viscosity &
density of the liquid & desired machine speed.
 The tube must freely enter the neck of the container & deliver the liquid deep enough
to permit air to escape without sweeping the entering liquid into the neck or out of the
container.
 To reduce the back flow of liquid, the tube should have the max possible diameter.
Excessive delivery force causes splashing & foaming.
 Filling machines should be designed in such way that liquid flows can be easily
demounted for cleaning & sterilization.
 Material of construction-non-reactive materials - borosilicate glass/SS.
 Syringes are usually made of SS, more pressure required for delivery of viscous liquid.
 For large volumes glass syringes are unsafe.
 Filling & Sealing of Parenterals
 The pressure pump filler often is operated semi automatically & differs from gravity
filler principally in that the liquid is under pressure. It is usually equipped with an
overflow tube connected to a receiver to prevent excess filling of the container.
 Vacuum filling is commonly used in tester filling lines for large volume of liquids
because it is more adaptable to automation. A vacuum is produced in a bottle when a
nozzle gasket makes a seal against the lip of the bottle to be filled.

Filling equipment for solids –

 Sterile solids such as antibiotics are more difficult to subdivide accurately & precisely
into individual dose containers than are liquids. The rate of flow of solid material tends
to be slow & irregular, particularly if finely powdered. Small, granular particles flow
must evenly.
 Filling & Sealing of Parenterals
 Manual filling- Sterile solids can be subdivided into containers by individual weighing
in a weighing balance with the help of small size SS scoop.
 Auger filling by automated filling machine- delivering measured quantities of free-
flowing material through funnel shaped hopper. The size & rotation of auger can be
adjusted to deliver a regulated volume of granular material from the funnel stem into
the container.
 Vacuum filling machine - an adjustable cavity in the rim of the filling wheel is filled by
vacuum as the wheel passes under the hopper. The contents are held by vacuum until
the cavity is inverted over the container, when a jet of sterile air discharge the dry
solids. This machine is suitable for poor or less flow property solid.
 Sealing of parenteral products – Containers should be sealed in the aseptic area
immediately adjacent to the filling machine. Sealing of containers assures the users
that it has not been opened.
 Filling & Sealing of Parenterals
Sealing of ampoules –
 Closed by melting a portion of the glass of neck to form either bead seals (tip-seals) or
pull seals.
 Tip seals are made by melting sufficient glass at the tip of ampoule neck to form a bead
of glass & close the opening.
 Pull-seals are made by heating the neck of rotating ampoule below the tip, then pulling
the tip away to form a small, twisted capillary just prior to being melt closed.
 The heating with a high temperature gas oxygen flame must be even & carefully
controlled to avoid gas distortion of the seal.
 Excessive heating of air & gases in the neck causes expansion against the soft glass with
the formation of fragile bubbles at the point of seal.
 Pull sealing is a slower process & more reliable than tip sealing. Powder ampoules or
other types having a wide opening must be sealed by pull-sealing.
 Sometimes ,displacement of air-to prevent decomposition. This is done by introducing
a stream of inert gas (N2 or CO2) during/after filling with the product.
 Filling & Sealing of Parenterals
Sealing of ampoules, Sealing bottles, cartridges & vials –
 The surface of the rubber closure is usually coated with silicone to
reduce friction .
 Aluminum caps are used to hold rubber closures in place. Single caps
may have a permanent center hole or a center that is torn away at the
time of use to expose the rubber closure.
 Double aluminum caps usually have a inner cap with a permanent
center hole which in use is exposed when the entire outer cap is torn off.
 The triple aluminum caps are used for large bottles with rubber closures
having permanent holes for attachment to administration sets.
 Lyophilisation
Lyophilisation (freeze dried products) –
o Solutions intended to be freeze dried must be aqueous
o Drying process involves removal of water by sublimation.
o Final product-Solid residue (cake) after drying - reconstitution at the time of use.
o Added substances required in the formulation must not be volatile under the
conditions of drying, therefore antibacterial agents such as phenol, chlorobutanol &
benzyl alcohol must not be used

Fundamental process steps are:

o Freezing: the product is frozen. This provides a necessary condition for low
temperature
o Vacuum: after freezing, the product is placed under vacuum. This enables the frozen
solvent in the product to vaporize without passing through liquid phase, a process
known as SUBLIMATION.
 Lyophilisation
o Heat: Heat is applied to the frozen product to accelerate sublimation.
o Condensation: Low-temperature condenser plates remove the vaporized solvent
from the vacuum chamber by converting it back to a solid.

o Freeze Drying
• Freezing the product solution to a temperature below its eutectic temperature.
• Decrease the shelf temperature to -50°C.
• Low temperature and low atmospheric pressure are maintained.
• Freons are used as refrigerant
• Formation of ice crystals occurs.
• The rate of ice crystallization define the freezing process and efficiency of primary
drying
 Lyophilisation
o Primary Drying (Sublimation)
• Heat is introduced from shelf to the product under graded control by electrical
resistance coils or circulating silicone.
• The temperature and pressure should be below the triple point of water i.e., 0.0098°C
and 4.58mmHg.
• The driving force is vapor pressure difference between the evaporating surface and the
condenser.
• Easily removes moisture up to 98% to 99%.

o Secondary Drying (Desorption)

• The temperature is raised to 50°C – 60°C and vacuum is lowered about 50mmHg.
• Bound water is removed.
• Rate of drying is low.
• It takes about 10-20 hrs.
 Lyophilisation
o Packing
• After drying the vacuum is replaced by filtered dry air or nitrogen to establish atmospheric
pressure
• Ampoules are sealed by either tip sealing or pull sealing method
• Vials and bottles are sealed with rubber closures and aluminum caps
o Common Lyophilized Products Pharmaceuticals –
*large and small molecules *Bacteria
*Viruses *Vaccines
*Plasma *Small zoological specimens

o Applications Pharmaceutical and biotechnology –

*to increase the shelf life of products, such as vaccines and other injectables
o Food industry
* to preserve food. * to produce flavouring agents.
*freeze-dried fruits. * Instant coffee powder.
 Lyophilisation
o Advantages of Lyophilization
• Removal of water at low temperature
• Thermolabile materials can be dried.
• Compatible with aseptic operations
• More precise fill weight control
• Sterility can be maintained.
• Reconstitution is easy
o Disadvantages of Lyophilization
• Many biological molecules are damaged by the stress or/and/both freezing.
• The product is prone to oxidation, due to high porosity and large surface area.
Therefore the product should be packed in vacuum or inert gas.
• Costly
• Long time process
 LVP MANUFACTURING
• DISPENSING (Requisition, Potency Calculation)
• GARMENTS & ACCESSORIES STERILIZATION
• CIP OF PROCESSING VESSEL AND TRANSFER LINE
• PRODUCT FILTER INTEGRITY TEST (Before Filtration)
• SIP OF PROCESSING VESSEL AND TRANSFER LINE
• MANUFACUTRING (Compounding) Example: Batch size 5000 L
• Dispense 3000 L Water For Injection into the compounding vessel & cool up to 25-35˚C
temperature
• Start stirring at 1000-1400 rpm for 5 Minutes, Add the Sodiaum Chloride BP into
compounding vessel and stir to disperse completely ,Stirring until completely dissolve
• Add the Potassium Chloride Chloride BP, Calcium Chloride Dihydrate BP ,Sodium
Lactate 50% Solution USP (one after another) Stirring until completely dissolve
• Final volume adjustment upto 5000 L, Make the final volume up to 5000L with WFI
while stirring (1200 to1400rpm). Mix the bulk solution after volume adjustment for 10
to 15 minutes.
 LVP MANUFACTURING
• pH adjustment: If required  
• Adjust the pH (6.20 – 6.50) by adding the Lactic Acid 90%, Pyrogen Free/ Sodium
Hydroxide, Injectable Grade solution into compounding vessel step by step & record.
• IN PROCESS CHECK
• Check & record the appearance & pH of the solution.(Appearance: A clear solution free
from any visible foreign particles, pH: 6.00 – 7.50)
• SOLUTION SAMPLING
• PRODUCT FILTERATION
• PRODUCT FILTER INTEGRITY TEST (After filtration) 
• Perform forward flow test after finish the solution. Attach print out of integrity tester
with batch documents.
• COMPOUNDING RECONCILIATION
• BFS MACHINE
• Bring received Insocaps, pp granules, take the materials into granules Bin
 Essential Elements
• Facility
– Design
Zoning
Differential Pressure
Temperature
Relative Humidity
Personnel and Material Flow
– Air Filtration
• Equipment
– Material of Construction
– Sanitization
– Component Preparation/Sterilization
 Essential Elements
• Process
– Product Formulation
– Filtration
– Filling
– Lyophilization
– Capping
• Personnel
– Gowning Qualification
– Aseptic Technique
• Control and Verification
– Environmental and Personnel Monitoring
– Aseptic Filling Simulations (Media Fills)
 Essential Elements
• Finished Product Testing
– Sterility Testing
– Particulate Testing
– Container Closure Integrity Testing
– Other Final Product/Release Testing
– Stability Testing
• Documentation
– Media Fill Records
– Production Batch Records
– EM Trend Data
– Release Testing Batch Records
– Investigation
• Corrective Actions
 Environmental Monitoring Components

• Airborne nonviable particulate monitoring

• Airborne viable contaminant monitoring
• Viable contaminant monitoring of surfaces
• Viable contaminant monitoring of personnel
• Temperature and humidity monitoring
• Pressure differential monitoring
• Water monitoring
 Sterile Production
Manufacture of sterile preparations
• Manufacturing operation -appropriate environment cleanliness level.
• Minimize risks – particulate and microbiological contamination – product
and material.
• Different class of sterile pharmaceutical preparations:
 Grade A
– Local zone, high risk operations, e.g. filling, aseptic connections
– Usually UDAF systems used
 Grade B
– Background environment to grade A (in case of aseptic preparation and
filling)
 Grade C and Grade D
– Clean areas for less critical operations
 Sterile Production
Manufacture of sterile preparations
• To reach Grade B, C and D, the number of air changes should be appropriate
to the size of the area, number of personnel, equipment present.
• Minimum of 20 air changes per hour.
• Clean up time about 15-20 minutes.
• Good airflow pattern in the area & HEPA-filtered air.
• Suitable methods to determine particulate matter and micro- e.g. EU, ISO,
USA.
• Control particulate during operation & Monitoring during operation.
• Alert and action limits for particulate and micro & Action taken when
exceeded.
• Area grades should be proven (e.g. validation runs, media fills, environment,
time limits – based on microbiological contamination found)
 Sterile Production
General Considerations
• Minimizing risks of contamination – microbiological, particulate
matter, pyrogen.
• Production in clean areas & appropriate standard of cleanliness
(Premises).
• Filtered air supplied.
• Airlocks for entry - Personnel ,equipment & Materials
• Separate areas for operations
– component preparation (containers and closures)
– product preparation, filling, sterilization, etc.
 Sterile Production
Equipment
• Effective sterilization of equipment.
• Maintenance and repairs from outside the clean area.
• Planned maintenance, validation and monitoring.
– Equipment, air filtration systems, sterilizers, water treatment
systems.
• Water treatment plants and distribution system
– Design, construction, maintenance.
• Water for Injection (WFI)
– Produced, stored, distributed – prevention of growth of
microorganisms.
– Constant circulation at temperature above 70ºC.
 Sterile Production
Premises
• Design
– Avoid unnecessary entry of supervisors and control personnel.
– Operations observed from outside.
• In clean areas, all exposed surfaces:
– Smooth, impervious, unbroken.
– Minimize shedding and accumulation of particles, microorganisms.
– Permit cleaning and disinfection.
– No uncleanable recesses, ledges, shelves, cupboards, equipment.
 Sterile Production
Premises(2)
– Sliding doors undesirable
– False ceilings sealed
• Changing rooms
– Designed as airlocks & Interlocking system for doors
– Separate rooms for entry and exit desirable
– Hand washing facilities
– Visual and/or audible warning system
– Use filtered air supply to maintain pressure cascade
• Free from Pathogenic, highly toxic materials
 Sterile Production
Premises(3)
• Pressure cascade may be different, approximately 10 to 15 Pascales
• Decontamination procedures – air, equipment, garments
• Warning system to indicate failure in air supply
• Pressure differential – results regularly recorded
• In clean areas, all exposed surfaces :
– Proper installation of pipes and ducts, no recesses, no unsealed
openings
– Sinks and drains avoided, and excluded in Grade A and B areas
 Sterile Production
Sanitation
• Frequent cleaning of areas & monitoring of clean areas.
• Regular monitoring of microorganisms.
• Monitoring of disinfectants and detergents.
• Monitoring of personnel and surfaces after critical operations.
• High standards of hygiene and cleanliness.
• Periodic health checks.
• Frequent monitoring in areas
– Settle plates, volumetric air samples, surface sampling (swabs and
contact plates).
• Results considered when batch release is done.
 Sterile Production
Personnel
• Minimum number of personnel in clean areas.
– Especially during aseptic processing.
• Inspections and controls from outside.
• Training to all including cleaner and maintenance staff
• No shedding of particles.
• No introduction of microbiological hazards.
• Clothing of appropriate quality:
• Hair, beard, moustache covered.
• Protective clothing and shoes.
• Single or 2-piece suit (covering wrists, high neck), shoes
• Headgear, beard and moustache covered, masks, gloves.
• No shedding of fibres, and retain particles shed by operators
 Sterile Production
Personnel(2)
• Change at every working session, or once a day.
• Change gloves and masks at every working session.
• Disinfect gloves during operations.
• Washing of garments – separate laundry facility.
• No damage.
• No outdoor clothing.
• Changing and washing procedure.
• No watches, jewellery and cosmetics.
 Sterile Production
Environmental Monitoring
Microbiological
• Air samples
• Surface swabs
• Personnel swabs
Physical
• Particulate matter
• Differential pressures
• Air changes, airflow patterns
• Clean-up time/recovery
• Filter integrity
• Temperature and relative humidity
• Airflow velocity
 Sterile Production
Processing
• Water sources, water treatment systems and treated water
• Monitored regularly –Chemicals -Biological contamination -Endotoxin
• Records of results and action taken
• Starting materials – microbiological contamination should be minimal
• Components, bulk product containers and equipment
– No fibre generation
– Sterilized when used in aseptic areas
• Used in clean areas, passed through double-ended sterilizers or use triple
wrapping
• Gas used to purge solution or blanket a product – passed through a
sterilizing filter