Session - 3

“Controlling the Cleanroom

Working Environment”
(1h 30m)

Sumant Baukhandi
Pharma Institute of GMPs

06/11/22 1
 Points Covered
 Defining an Aseptic State
 Attaining an Aseptic State – Quality By Design
 Apply Risk Based Approach
 People & Aseptic Technique
 Complexity of Aseptic Processing
Approaches
 Sanitization Techniques in Cleanrooms

06/11/22 2
 Aseptic State
 Defining an Aseptic State
- What is Asepsis?
- Is it sterile?
 Let us see!!

06/11/22 3
 Aseptic State
 Defining an Aseptic State
1. Sepsis: Comes from Greek for decay or
putrid. Indicates bacterial contamination.
2. Asepsis: Absence of significant
contamination.
3. Aseptic techniques are used to prevent
contamination of products, operating
personnel, and processing areas &
equipment

06/11/22 4
 Aseptic State
 Aseptic Processing - A mode of processing
pharmaceutical and medical products that
involves the separate sterilization of the
product and of the package
(containers/closures or packaging material
for medical devices) and the transfer of the
product into the container and its closure
under microbiologically critical controlled
conditions.

06/11/22 5
 Aseptic State
 Clean Room - A room in which the concentration of
airborne particles is controlled to meet a specified
airborne particulate Cleanliness Class.
 In addition, the concentration of microorganisms in the
environment is monitored
 Each Cleanliness Class defined is also assigned a
microbial level for air, surface, and personnel gear.
 Controlled Environment - Any area in an aseptic process
system for which airborne particulate and microorganism
levels are controlled to specific levels, appropriate to the
activities conducted within that environment.

06/11/22 6
 Aseptic State
 Defining an Aseptic State
1. Sterility - Within the strictest definition of sterility,
an article is deemed sterile when there is complete absence
of viable microorganisms.
Absolute sterility cannot be practically demonstrated
without testing every article in a batch.
Sterility is defined in probabilistic terms, where the
likelihood of a contaminated article is acceptably remote.

06/11/22 7
 Control of Microbial Growth:
Rate of Microbial Death
When bacterial populations are heated or treated
with anti-microbial chemicals, they usually die at a
constant rate.

06/11/22 8
 Aseptic State
 How to Attain an Aseptic State?
 Quality By Design
 Facility Design
- Building & Facilities
- HVAC Systems
- Man - Material Flow
- Behavior of Operating Personnel
- Sanitation, Sanitization, Disinfection techniques
- Sterilization processes / Filtration process
- Handling of material / components
06/11/22 9
 Aseptic State
 People & Aseptic Technique
 General Hygiene
 General Awareness on Microbiology & Contamination
 Know your Area classification regime and restricted
access
 Know your gowning regime
 Knowing the Aseptic processes – how to handle?
 Know the Environmental conditions inside that directly
influence contamination & growth
 Know the sources of contamination & controls
 Know the sterilization systems & processes
06/11/22 10
 Aseptic State
 People & Aseptic Technique
 Skills: Attitude, temperament, diligence, thoroughness
 What is Aseptic processing & how does it work?
 Why is aseptic filling performed?
 Aseptic connections, transfers,
 Quality Systems: Validation, Calibration, Training,
Documentation, Change control
 Why we do microbiological monitoring & testing?

06/11/22 11
 Aseptic State
 People & Aseptic Technique
 How to move & behave in a manufacturing environment?
 How to handle materials / components / machines?
 How to intervene a process appropriately?
 How to perform sanitation, sanitization, sterilization, filtration?
 Good Apparel, Attire,
 Proper gowning practice, gloving,
 Good Clean room behavior
 Good Laminar airflow, HEPA Filtration
 Tacky mats
 Good Hand washing & disinfection practices
 Good use of cleaners, mops, swipes, swabs, RODAC plates, IPA

06/11/22 12
 Aseptic State
 Complexity of Aseptic Approaches
 Powder in Vials
 Solution in Vials
 Glass Ampoules & Sterilization
 Lyophilization processes – glass vials
 Opthalmic solutions
 Nasal Drops
 Bulk APIs – regular & lyophilized – equipment
complexities
 Risks to identified, assessed, minimized
 Equipment cleaning & SIPs
 Aseptic Process Filtrations
06/11/22 13
 Sanitization Techniques
 Sanitization Techniques in Cleanrooms
 Sanitization is a complex technology, not
well recognized in pharmaceutical field
 Some important terms:
1. Antimicrobial: This is an agent intended
for minimizing growth or destroying
microorganisms in vitro (sanitizers,
germicides, disinfectants, sterilizers &
microbiostats)
06/11/22 14
 Sanitization Techniques
 Sanitization Techniques in Cleanrooms
2. Sanitizer: It is a chemical agent that
specifically kills microbial contamination in
the form of vegetative cells
3. Sterilizer: This may be defined as a
chemical agent that destroys or eliminates
all forms of microbial life in an inanimate
environment. Sanitizers are not necessarily
sterilizers
06/11/22 15
 Sanitization Techniques
 Sanitization Techniques in Cleanrooms
4. Disinfectant: This is an anti-microbial
agent employed to destroy pathogens on
inanimate objects
5. Microbiocides: These agents kill
microorganisms (bactericide, fungicide,
sporicide, virucide)
6. Microbiostats: These agents only inhibit
growth of microorgnisms
06/11/22 16
 Sanitization Techniques
 Selection of Agent
 No single sanitizer achieves the ideal goal.
Following should be the considerations:
- Kills a wide range of microbes
- is non-toxic to humans
- is non-corrosive & non-staining to equipment
- possesses detergent action
- has stability
- has quick action
- will not be inactivated by organic matter
- produces desired residual action, and
- is inexpensive
06/11/22 17
 Sanitization Techniques
 Assessing Scope of Application
 The microbiologist must select a single agent or a
combination, governed by the standards of her/his
company,
that produces maximum effectiveness at a
minimum cost
 Considerable research & evaluation is required to
determine the preferred sanitizer

06/11/22 18
 Sanitization Techniques
 Assessing Scope of Application
 Know the surfaces to be sanitized:
 Inside or outside: easily accessible
 Compatibility of the surfaces
(glass, ceramics, stainless steel are resistant)
 Type of finish: porous, smooth, rough, grainy
(for porous – add surfactant to aid penetration)
 Method of Application is another aspect:
 Spraying,
 Mopping,
 Fogging,
 Submerging
(which is more appropriate?)
 What should be the contact time?

06/11/22 19
 Sanitization Techniques
 Rotation of Sanitizer(s)
 Since no single sanitizer kills all types of
microbes, it is prudent to rotate sanitizers
 Rotation frequency to be determined
 Weekly or monthly rotation
However, rotation should be done only if deemed
necessary (when specific data indicates a need)

06/11/22 20
 Sanitization Techniques
 Selection of Sanitizer(s)
 Merits of four major groups should be considered
 Chlorinated compounds: most widely used & economical
- Cl2 solutions in water – form hypochlorous acid
- Chloramines & Cl2 gas - increasing concentrations kills bacteria
- Hypochlorite solutions – increasing concentrations decreases
their power to kill
 Iodine compounds – Iodophors contain solubilizing agent in I2
 Quaternary ammonium compounds – sanitize at low
concentrations, and
 Acid-anionic compounds – combinations of organic or inorganic
acids & surface-active agents (H2PO4 & alkyl sulfonate group)

06/11/22 21
06/11/22 22
 Sanitization Techniques
 Selection of Sanitizer(s)
1. Phenols and Phenolics:
 Phenol (carbolic acid) was first used by Lister as a disinfectant.
 Rarely used today because it is a skin irritant and has strong odor.
 Used in some throat sprays and lozenges.
 Acts as local anesthetic.
 Phenolics are chemical derivatives of phenol
 Cresols: Derived from coal tar (Lysol).
 Biphenols (pHisoHex): Effective against gram-positive
staphylococci and streptococci. Used in nurseries. Excessive use
in infants may cause neurological damage.
 Destroy plasma membranes and denature proteins.
 Advantages: Stable, persist for long times after applied, and remain
active in the presence of organic compounds.

06/11/22 23
 Sanitization Techniques
 Selection of Sanitizer(s)
2. Halogens: Effective alone or in compounds.
A. Iodine:
 Tincture of iodine (alcohol solution) was one of first antiseptics used.
 Combines with amino acid tyrosine in proteins and denatures
proteins.
 Stains skin and clothes, somewhat irritating.
 Iodophors: Compounds with iodine that are slow releasing, take
several minutes to act. Used as skin antiseptic in surgery. Not
effective against bacterial endospores.
 Betadine
 Isodine

06/11/22 24
 Sanitization Techniques
 Selection of Sanitizer(s)
2. Halogens: Effective alone or in compounds.
B. Chlorine:
 When mixed in water forms hypochlorous acid:
Cl2 + H2O ------> H+ + Cl- + HOCl
(Hypochlorous acid)
 Used to disinfect drinking water, pools, and sewage.
 Chlorine is easily inactivated by organic materials.
 Sodium hypochlorite (NaOCl): Is active ingredient of
bleach.
 Chloramines: Consist of chlorine and ammonia. Less
effective as germicides.
06/11/22 25
 Sanitization Techniques
 Selection of Sanitizer(s)
3. Alcohols:
 Kill bacteria, fungi, but not endospores or naked viruses.
 Act by denaturing proteins and disrupting cell membranes.
 Evaporate, leaving no residue.
 Used to mechanically wipe microbes off skin before
injections or blood drawing.
 Not good for open wounds, because cause proteins to
coagulate.
 Ethanol: Drinking alcohol. Optimum concentration is 70%.
 Isopropanol: Rubbing alcohol. Better disinfectant than ethanol.
Also cheaper and less volatile.

06/11/22 26
 Sanitization Techniques
 Selection of Sanitizer(s)
4. Heavy Metals:
 Include copper, selenium, mercury, silver, and zinc.
 Oligodynamic action: Very tiny amounts are effective.
A. Silver:
 1% silver nitrate used to protect infants against gonorrheal eye
infections until recently.
B. Mercury
 Organic mercury compounds like merthiolate and mercurochrome
are used to disinfect skin wounds.
C. Copper
 Copper sulfate is used to kill algae in pools and fish tanks.
06/11/22 27
 Sanitization Techniques
 Selection of Sanitizer(s)
5. Quaternary Ammonium Compounds (Quats):
 Widely used surface active agents.
 Cationic (positively charge) detergents.
 Effective against gram positive bacteria, less effective against gram-
negative bacteria.
 Also destroy fungi, amoebas, and enveloped viruses.
 Zephiran, Cepacol, also found in our lab spray bottles.
 Pseudomonas strains that are resistant and can grow in presence of
Quats are a big concern in hospitals.
 Advantages: Strong anti-microbial action, colorless, odorless,
tasteless, stable, and nontoxic.
 Disadvantages: Form foam. Organic matter interferes with
effectiveness. Neutralized by soaps and anionic detergents.

06/11/22 28
 Sanitization Techniques
 Selection of Sanitizer(s)
6. Aldehydes:
 Include some of the most effective anti-microbials.
 Inactivate proteins by forming covalent cross-links with
several functional groups.
A. Formaldehyde gas:
 Excellent disinfectant.
 Commonly used as formalin, a 37% aqueous solution.
 Formalin was used extensively to preserve biological
specimens and inactivate viruses and bacteria in vaccines.
 Irritates mucous membranes, strong odor
 Also used in mortuaries for embalming.

06/11/22 29
 Sanitization Techniques
 Selection of Sanitizer(s)
6. Aldehydes:
B. Glutaraldehyde:
 Less irritating and more effective than formaldehyde.
 One of the few chemical disinfectants that is a sterilizing
agent.
 A 2% solution of glutaraldehyde (Cidex) is:
 Bactericidal, tuberculocidal, and viricidal in 10 minutes.
 Sporicidal in 3 to 10 hours.
 Commonly used to disinfect hospital instruments.
 Also used in mortuaries for embalming.
06/11/22 30
 Sanitization Techniques
 Selection of Sanitizer(s)
7. Gaseous Sterilizers:
 Chemicals that sterilize in a chamber similar to an autoclave.
 Denature proteins, by replacing functional groups with alkyl groups.
A. Ethylene Oxide:
 Kills all microbes and endospores, but requires exposure of 4 to 18
hours.
 Toxic and explosive in pure form.
 Highly penetrating.
 Most hospitals have ethylene oxide chambers to sterilize mattresses
and large equipment.

06/11/22 31
 Sanitization Techniques
 Selection of Sanitizer(s)
8. Peroxygens (Oxidizing Agents):
 Oxidize cellular components of treated microbes.
 Disrupt membranes and proteins.
A. Ozone:
 Used along with chlorine to disinfect water.
 Helps neutralize unpleasant tastes and odors.
 More effective killing agent than chlorine, but less stable and more
expensive.
 Highly reactive form of oxygen.
 Made by exposing oxygen to electricity or UV light.

06/11/22 32
 Sanitization Techniques
 Selection of Sanitizer(s)
8. Peroxygens (Oxidizing Agents):
B. Hydrogen Peroxide:
 Used as an antiseptic.
 Not good for open wounds because quickly broken down
by catalase present in human cells.
 Effective in disinfection of inanimate objects.
 Sporicidal at higher temperatures.
 Used by food industry and to disinfect contact lenses.
C. Benzoyl Peroxide:
 Used in acne medications.

06/11/22 33
 Sanitization Techniques
 Selection of Sanitizer(s)
8. Peroxygens (Oxidizing Agents):
D. Peracetic Acid:
 One of the most effective liquid sporicides available.
 Sterilant :
 Kills bacteria and fungi in less than 5 minutes.
 Kills endospores and viruses within 30 minutes.
 Used widely in disinfection of food and medical
instruments because it does not leave toxic residues.

06/11/22 34
 Efficiency of Different Chemical
Antimicrobial Agents

06/11/22 35
 THANKS!
For inquiries please contact –

Sumant Baukhandi, PhD

 
Director – Pharma Institute of GMPs,
Director – pharmaACCESS

7, Phase-1, Vasant Vihar,

Dehradun-246 006
Uttaranchal State, INDIA
 
Landphone: 91-135 276 3092
Cellphone: 91- 99970 10901
Email ID: pigmpinstt@gmail.com ;
sumant091154@rediffmail.com

Website: www.pigmp.com

06/11/22 36