Contamination Control in Sterile Production

Question 1

Non-sterile product

Answer 1

• Limited Microbial Burden
• Absence of objectionable organisms e.g. E.Coli, S.A, Samonella
• Oral/ topical products
• Otic, nasal, vaginal and rectal

Question 2

Sterile Product

Answer 2

• FREE from m.o, pyrogens, particulates
• safe, stable and therapeutically active
• acceptable quality for patient administration
  Eg Injectables, opthalmic prep, otic (post surgical), irrigation, dialysis, TPN, medical devices

Question 3

Sterilisation

Answer 3

• Process intended to kill or remove all m.o
• With an acceptably low probability of 1 in million chance of one m.o surviving
• depends on contamination control and sterility assurance procedures and practices
• knowledge, skills and attitude of staff

Question 4

Contamination control of the area

Answer 4

• segregated
• limited authorised entry
• operational arrangement
• sterile production clean rooms
• maintenance and monitoring
• cGMP

Question 5

Ante Room

Answer 5

• Pre-room entry into graded cleanrooms
• Sticky mat/ floor
• Gowning/ hand washing stations
• Filtered air (HEPA filter)
• Laminar air flow
• Positive air pressure

Question 6

Clean rooms

Answer 6

• Positive air pressure
• Filtered air supply (HEPA)
• Constant flushing of room with clean air
• smooth impervious surface

Question 7

Laminar Flow Cabinet

Answer 7

• 3.5 Clean room in class 350 Clean room
• Double barrier system with high pressure and HEPA
• 2 types: horizontal and vertical

Question 8

Horizontal Flow Cabinet

Answer 8

• HEPA laminar air flow
• unidirectional horizontal air flow
• protect product from operator
• prepare non-cytotoxic products
• Work bench divided into 3 zones:
  Inner - set up sterile work items, avoid shadowing of critical areas e.g. syringe
  Middle - Aseptic assembly
  Outer - Finished and sealed product

Question 9

Down Flow Cabinet

Answer 9

• Aseptic prep for cytotoxic products
• HEPA filtered vertical air flow
• Product product and operator
• Has glass shield cover
• Double gloving and owning
• Charcoal filtered air outlet
• Negative pressure than the cytotoxic ante room (air lock)

Question 10

Isolator Cabinet

Answer 10

• almost complete separation between product, operator and surrounding work environment
• use half suit or glove ports to a sterilised isolator chamber

Question 11

Monitoring LFC

Answer 11

1. Air flow tester
   - used to show air currents in the LFC
   - use a tube with sulphuric acid to generate white aerosol smoke to show air flow pattern
2. Smoke test for HEPA filter leakage
   - Dioctyl phthalate particles which are 0.5 micron particle size
3. Microbiological monitoring
   - air, surface sampling using sterile and fertile nutrient agars

Question 12

Operator

Answer 12

• major source
• hand wash, gowning, aseptic techniques
• need training and checks, report medical conditions
• remove jewellery, cosmetics

Question 13

Sterile over garment

Answer 13

• specially packed and folded to minimise contamination
• non-shedding
• has indicator of sterilisation
• hair cover, gown, shoe covers, face masks
• systematic gowning

Question 14

Equipment and containers

Answer 14

• Can be source of contamination OR protect product from contamination
• Rinse in 350 clean room with pyrogen free distilled water
• Sterilise: use systematic wrapping, chemical indicators and sterility testing
• Container must not interact with fill, generate particles or pyrogens, retain ingredients from fill, not release substance into products, heat stable for heat sterilisation methods

Question 15

Sterile Manufacturing Methods

Answer 15

1. Terminal Sterilisation
2. Aseptic Filtration
3. BFS - Blow Fill Seal Technology

Question 16

Terminal Sterilisation

Answer 16

1. Filling and sealing in the LFC, filtration of the solution using 0.45um filter
2. Use heat steriliser to sterilise the finished product
3. Sterility testing of the finished product

Question 17

Aseptic Filtration

Answer 17

1. Using strict aseptic techniques in LFC e.g. water for inn, sterile equipment
2. Sterilisation by filtration using 0.22um filter
3. Sterility testing of finished product

Question 18

Water Control - contamination control

Answer 18

Tap
Deionised
Purified
Water for irrigation
Water for injection

Question 19

Deionised Water

Answer 19

Demineralised water by passing tap water through synthetic ion-exchange resin beds
Removes cations and anions
Does not remove bacteria
Used in cosmetics, topical formulations, dilution of disinfectants

Question 20

Purified Water

Answer 20

Processed by distillation
Apyrogenic
Free from microorganism as it leaves the still but may later be contaminated
Used for oral/ topical products, rinsing of equipment
Not for IV, parenteral

Question 21

Water for irrigation

Answer 21

Sterile and pyrogen free purified water sterilised using terminal heat sterilisation
Oral/ topical formulation
Irrigation of body cavities and wounds
Not for parenteral, IV

Question 22

Water for injection

Answer 22

Sterilised and filtered
Pyrogen Free
Used for injection, parenteral and eye drops
QC test: particulate contamination

Question 23

QC TEST

Answer 23

sterility, pyrogen, pH, conductivity, chemical composition

Question 24

Water Contaminants

Answer 24

Particulate matters
Microorganisms
Pyrogens
Endotoxins
Organic and Inorganic substances

Question 25

Particulate Contamination

Answer 25

Drug, dust, glass
Sterile solution must be kept FREE
Checks:
1. Coulter Counter
Voltage pulse is produced as particle passes through 2 electrodes, height of pulse is proportional to volume of particle, particles in solution is counted and sized per second
2. Light Obscuration
Particle obscures light during transit through laser beam
Electrical pulses are produced to size the particles
3. Microscopic Count
Membrane filtration using 0.45um filter of solution and microscopic examination of particles retained on the filter

Question 26

Microbial Contamination

Answer 26

• contamination in water supply, storage and distribution
• distillation and filtration to remove
• may have subsequent contamination, store distilled water at 65 4 hours prior to sterilisation within 4 hours
• contamination monitoring using membrane filtration method, then incubate filter to detect any microbial presence

Question 27

Pyrogen Contamination

Answer 27

• substances that can cause a pyrogenic feverish response when entered into blood or tissue
• heat resistant
• eg. bacterial endotoxins: Lipopolysaccharides from Gram negative bacteria cell wall, pyrogenic

Question 28

How to remove pyrogens from water

Answer 28

1. Heat: 250/ 45, 180/ 4hr
2. Chemical treatment: heating with strong alkali
3. Physical removal: Distillation, Reverse Osmosis
   Reverse Osmosis - Uses a semi-permeable membrane which retains molecules >300 MW, rejects pyrogens/ bacteria/ inorganic materials (85-95%), 10-50% fed water passes through membrane

Question 29

Pyrogen Testing

Answer 29

1. Rabbit testing
   Test solution injected into ear vein of rabbits, test rectal temp pre and post injection, solution pass test if increase in temp is <1.4
2. Limulus Amoebocyte Lysate (LAL) (bacterial endotoxin test)
   - LAL reagent mix with test solution. Reaction occurs and if endotoxin is present, results present: gel formation, change in turbidity, change in colour
   - MOA: LAL reagent contains pro-clotting enzymes, clotting proteins and CA2+, LAL enzyme recognises endotoxin (Reaction initiation), enzymes activated in a cascade (propagation), solution becomes turbid, proteinaceous clot develops and gel formation within 20 mins at 37 deg

Question 30

Characteristics of LAL test

Answer 30

• perform duplicate tests
• use negative and positive control
• containers must not adsorb or generate endotoxin

Limitations:
- Need to reconstitute lysate, product interference, pH and temp, sensitivity and limit of detection

Question 31

Quantitative method

Answer 31

Calibration curve: conc endotoxin vs reaction time
Turbidimetric kinetic: time needed for solution to become turbid
Chromogenic kinetic: time needed for liberation of colour dye
Automatic portable endotoxin test kit