STERILITY

Definition:
According to WHO1, Sterility can be defined as;
“The freedom from the presence of viable microorganisms”
However, the conditions that guarantee absolute sterility are usually too harsh for active ingredients, and the
definition of sterility for a medicinal product must be defined in functional terms.

1. Sterile Area in Pharmaceutical Industries:2

A sterile area, also called as controlled area, is an enclosed environment or room with a flawless control over
particulate contamination. More specifically, the areas have a controlled contamination level, which is specified
regarding the number of particles for every cubic meter, for a specified particle size. The restricted areas are
constructed with impeccable humidity, temperature and pressure control to minimize the generation,
introduction, and retention of particulate matter inside the rooms.
All pharmaceutical products are manufactured in controlled areas where the environmental conditions are
controlled and product remains safe from temperature, humidity and contaminants during manufacturing.
Used for sterile pharmaceutical manufacturing, the controlled areas ensure low levels of environmental
pollutants, such as airborne microbes, dust, aerosol particles and chemical vapors. The classified rooms help the
sterile pharmaceutical industry to manufacture products that are free from particulate and microbial
contamination. Indeed, sterile pharmaceutical controlled rooms are clean rooms that are fitted with HEPA filters,
and dehumidifier systems to allow preparation of pharmaceutical products in a moisture-free and contamination-
free environments. Today, controlled area (classified area) and its use in sterile product manufacturing have
grown in leaps and bounds.

1.1 Objectives of Using Controlled area in Sterile Pharmaceutical Manufacturing:

In the sterile pharmaceutical industry, the core objective of using controlled rooms is preventing contamination
and ensuring that preparations are completely sterile. The rooms help to eliminate contamination with chemicals,
bacteria, electrostatic charges, particles, and fibers. Since, sterile pharmaceutical products should not introduce
undesirable particles, chemicals, and bacteria into the patients, the controlled area (classified area) and its use in
sterile product manufacturing have become a critical quality assurance step for every drug company. Similarly,
because most surfaces of controlled rooms are made of poor conductors, they can easily develop and retain
undesirable electrostatic charges. Hence, the surfaces are cleaned and controlled in such a fashion that the
charges can be conducted away as soon as they are formed.

1
https://www.who.int/biologicals/vaccines/sterility_testing/en/
2
https://www.pharmaguideline.com/2017/05/controlled-areas-in-sterile-manufacturing.html
1.2 Sections and Classification of Controlled area in Sterile Pharmaceutical Manufacturing:
A sterile pharmaceutical controlled area is principally divided into two sections: the general area and the critical
area.
The general area is the section of the rooms where the existence of contaminants will not directly influence the
sterility of products. The general area should, however, be properly cleaned and controlled to avoid the transfer
of contaminants into the critical area.
The critical area is the section around the production point, where any contaminants may gain direct access to the
preparations. The critical area is usually protected using localized Laminar-Flow workstations and clean
benches.

1.3 Ensuring Complete Sterilization:

To manufacture completely sterile products, various stages of product processing such as component
preparation, filling and product preparation should be performed in separate sections of the controlled area.
Hence, manufacturing operations should be divided into stage-wise sterilization (for every stage) and terminal
sterilization (for the completed preparations).
To achieve thorough stage-wise sterilization, the controlled room should allow for at-rest and in-operation states.
At-rest state means the production equipment in the classified room operates without the presence of personnel.
On the other hand, in-operation state means the manufacturing processes are run by a specified and limited
number of personnel.
Therefore, the sterile pharmaceutical company should grade its controlled production into four levels: A.B, C
and D. Grade A should be the zone for high-risk operations such as the filling zone and the zone for making
aseptic connections, open vials and ampoules.
The zone should have laminar airflow systems with homogenous air speeds. Grade B should be the background
environment for grade A, allowing for aseptic preparation and filling. Grade C and D should be classified areas
that handle the less critical stages of sterile product manufacturing.
2. Sterile Area (Cleanroom) Qualification:3
Sterile area validation has different tests like air supply, air velocity, air changes, flow pattern, filter integrity,
pressure test, particle count, temperature, recovery test, microbial count, relative humidity, noise level and
vibration test.
Sterile area qualification and HVAC system qualification are two different things but most of the tests are same
because HVAC system is qualified by its performance as the quality of the area. Qualification document of the
HVAC system and the sterile area shuld be prepared separately.

The sterile area can be qualified by applying following tests:

2.1 Air supply capacity:
The purpose of this test to demonstrate that the air system is balanced and capable of delivering sufficient air
volumes (as per design) to maintain required air change (NLT 20) in the defined area. The air capacity will be
demonstrated by following the procedure of:
 Measure airflow in supply and returned duct
 Air volume to meet the designed required

2.2 Air velocity/Uniformity:

The purpose of this test to demonstrate that the air system is balanced and capable of delivering sufficient air
volumes to maintained a minimum cross-section velocity under HEPA terminal filter modules. This test also
meant to verify air velocities before the air encounters an obstruction as well as to verify horizontal/vertical air
velocity components at the point the air reaches an obstacle or a surface one-meter above the floor. The air
velocity/Uniformity will be demonstrated by following the procedure of:
 Measure air velocity at the filter face using a calibrated anemometer.\
 Measure at numerous sites to provide one measurement for every 0.37 m2 filter area
 For LAF air flows uniformity to be 0.45 m/sec ± 20%
 For other filters, airflow will be checked and ensure that airflow shall not exceed filter rating and
uniformity shall not exceed designed specification

2.3 Air change:

There is no minimum GMP requirement for air changes per hour. Airflow into and out of a space should be
based on providing the required cooling, heating, relative humidity, pressurization, particulate control, dilution
ventilation, recovery time from an upset (spill or dust emission) condition. These factors generally result in air
change rates of between 4 and 20. However, higher rates have also been used successfully. An adequate number
of fresh air quantity (as per designed) will demonstrate at the time qualification of the HVAC facility.
2.4 Air Flow Pattern:
To determine the airflow interaction with machinery and equipment, in the critical area protected by a
unidirectional flow the clean air system in the sterile area. This test determines the airflow pattern at the Rest
Test Phase to check the airflow pattern from clean area to less clean area. This test shall be demonstrated by:
 Visualize the air patterns at numerous points in the room using smock generate or Titanium Tetrachloride
sticks.
 Smock should flow from “clean” area to “less clean” area quickly and smoothly.
 To check the interference due to turbulence eddies in unidirectional airflow area, like sampling booth,
dispensing booth and under laminar airflow in the microbiological area
 This test is used to check that there is no the dead space in the critical area

2.5 HEPA Filter Integrity Test:

All HEPA filter installed in the facility will test for filter integrity test and filter leak test at the At-Rest Phase by
using PAO (Poly-alpha-olefin) aerosol into supply duct to the HEPA filter. The following test will be allowed to
demonstrate the filter integrity and leak test.
Sample stream challenge with a photometer and set the instrument for this challenge:
 Linear readout : 100% deflection
 Logarithmic readout: 1 x 104
To check the filter surface and seals with photometer for leak test to
 Linear ; < 0.01%
 Logarithmic: One division
Alternatively
 Measure the “normal” upstream particulate challenge to HEPA filter using particle counter
 Scan the filter face and seals with particle counter for the leak test
 >10
 -4 of upstream
 Not more than 3% area of the filter should not be repaired by the sealant

2.6 Pressurization Test:

There is no quantified (numerical) requirement for relative pressurization (accept microbiological lab in QC
area). The velocity and direction of airflow between spaces should be adequate to reduce the counter flow of
airborne particulates or vapor contaminants for spaces where airborne cross contamination is a concern.
In general, relative pressurization shall be set up to reduce airborne particulates and vapors from passing from an
open Level 3 Protection processing space to another incompatible Level 3 Protection space. Conversely,
pressurization should be set up to reduce airborne particulates from passing from the outdoors, above ceilings,
mechanical or similar spaces and from Level 1 Protection spaces to Level 3 Protection processing spaces.
Airlocks or buffer zones are used to separate production areas from adjacent common corridor/staging areas,
non- classified areas.
To provide protection, when the doors are closed, room pressure shall be demonstrably positive or negative.
Airlocks or buffer zones will provide additional protection if only one door is open at a time. Pressured airlocks
may have either positive or negative relative pressure, depending on what is best for the particular situation.
Airflow variations from dust collecting, vacuum or process systems and their effect on space pressurization shall
be demonstrated for in the operation of the HVAC system.
Prior to air balancing, rooms shall be inspected for obvious leakage and for architectural integrity. Leakage may
have a significant effect on the room air balance and/or upon the ability for particulates to enter or leave the
space.

2.7 Unidirectional airflow:

A unidirectional air flow (wherever applicable) will be demonstrated to check the interference due to turbulence
airflow in the area that laminarity of the system (like dispensing booth, sampling booth) not disturbed. The
Unidirectional Air Flow will be demonstrated by the following test:
 Operate the HVAC system of the sterile area and release smoke into the unidirectional air stream at
selected sites
 Measure the deviation of smoke stream from vertical or horizontal over 90 cm from the flow path
 To demonstrate that deviation in parallelism airflow shall not greater than 14º

2.7 Particle count:

There are no particulate classification requirements for OSD facilities such as those that exist for aseptic
processing.
Cross-contamination can originate from both the internal environment and outside the OSD facility. In all air
handling systems, the filtration should be evaluated for the adequate assistance of outdoor particulates.
The HVAC system was designed to demonstrate the Class - D requirement At Rest or As-Built level and it will
demonstrate at time qualification activities of HVAC system. Select any suitable particle counter instrument and
set it at an air flow rate of 1 cubic feet per minute, carry out the particle count at minimum 10 different locations
in the room which are representative of the room for one minute at each location at 3 feet height from the floor.
It must cover the central location of personal traffic. Note down the number of particles of size equal to 0.5
microns and larger and number of particles of size equal to 5 microns and larger at each designated location.

2.8 Temperature:
Room temperature may be a critical parameter for both open and closed operations. Most products, materials and
processes can handle a wide range in temperature. However, the width of this range decreases as the exposure
time increases. For example, the contents of a large processing vessel would typically not change temperature
measurably during typical sampling activities. If product or material is stored or exposed for significant periods,
however, (such as in a non-temperature controlled blender) then significant effects may occur.
Personnel consideration in temperature consideration: The USP excursion limits for finished product storage are
59°F - 86°F (15°C - 30°C) with a customary CRT (Controlled Room Temperature) working environment of
68°F - 77°F (20°C - 25°C) and a maximum MRT (Mean Kinetic Temperature) of 77°F (25°C). Existing HVAC
system had designed for 24 ± 2°C to all critical area in manufacturing, warehouse and Quality Control
department.
Process related consideration in temperature specification: In some area due to the presence of heat generated
equipment the temperature efficiency will be demonstrated in the compliances of the designed criteria at the time
of the qualification exercise. In some product, in-processing temperature requirement shall be maintained and
demonstrated 18°C ± 2°C. Actual product temperature monitoring (whenever required) will be performed
instead of room temperature monitoring.
Exhaust Consideration in Temperature Specification: Wherever the exhaust of the large quantity of the air from
the critical area requires a large volume of makeup air to be introduced into the in-process area to replace it. This
replaces air shall be conditioned prior to introduction in the in process area.
Air Flow Pattern Consideration in Temperature Specifications: In the mixed airflow critical area, a pocket of
stagnant air may develop. Temperature gradients in excess of those specified can result, air inlet and outlet are
not properly spaced. Proper mixing of conditioned air with re-circulated air and effective airflow pattern is
required to demonstrate the specified zoning temperature.

2.9 Recovery test:

To determine the capabilities of the system to recover from internally generated from contamination within
reasonable elapsed time period. Recovery test will be demonstrated by following the test of:
 Measure the particle counts at room air return outlets and establish “At rest” level.
 Generate the smoke at air inlets to room until particle counts at outlet is high and constant and switch of
the smoke generator
 Record the particle counts each minute until counts return “At Rest” level
 Recovery time should be approximately 15-20 minutes (EU GMP guide Annex-1)

2.10 Microbiological Counts:

Testing will be performed for a period of one year routine production in accordance with the sampling plan.
Sampling will be performed in accordance with the procedures detailed in Validation Protocol for sterile area.
The testing regime for the Environmental Monitoring will be defined in Validation Protocol. Throughout the
Performance Qualification period regular project team meetings will be held to discuss the testing results. Should
any failures occur during the Operation Qualification stage additional samples will be taken to ascertain the
cause and extent of the contamination.
Corrective action will be instigated on a case-by-case basis.

2.11 Relative Humidity:

Room relative humidity (RH) may affect exposed product or materials that are sensitive to air moisture. RH
levels generally have negligible effect on aqueous product. However, liquid product can lose moisture to a low
humidity room over an extended period. HVAC system in sterile area had designed to maintain the humidity not
more than 55% Humidity level will be maintained wherever product comes in the direct contact of the product.
A cooling coil type of dehumidification will be applied for maintaining the humidity in the required area. Using
drain tray and pipeline to make assure not to stagnant water in AHU will drain the condensed water out.
Relative humidity will be checked by using calibrated humidity meter from different location of Classified,
Unclassified area as defined in the Validation Protocol for sterile area. Humidity should not more than defined
alert and action limits in validation protocol for sterile area.

2.12 Noise level:

High level of noise may be present in the facility due to operation of the variety of equipment like high static
exhaust fan, vacuum pump, centrifugal pump etc. OSHA requires hearing conversation program when personnel
noise exposure exceeds an eight hour time weighed average sound level of 85 dBa. HVAC system has designed
not to generate more than 70 dBa noises in critical area during its normal operation.
Noise level will be check by using calibrated Octave Band analyser or any other similar equipment and measure
the noise levels at defined location to confirm the that noise level is within limits as defined in the validation
protocol for sterile area.

2.13 Vibration:
The HVAC system of the facility has design to generate a minimum vibration during it full-scale operation.
Vibration level generate by HVAC system will be checked by using calibrated accelerometer or any other similar
equipment and measure the vibration levels at defined location to confirm the that vibration level is within limits
as defined in the validation protocol.