Initial Qualification in Sterile

Manufacturing: Annex 1 and ISO 14644 – a

comparison

Dr.-Ing. Jürgen Blattner

BSR Ingenieur-Büro

20. April 2021

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 Contents

1. Qualification measurements according Annex1

2. Qualification measurements according ISO
3. Measurement intervals
4. documentation

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 1. Measurements for qualification
(according to Annex1)
Ø Clean room classification*
Ø Installed filter leakage and integrity testing*
Ø Airflow measurement - Volume / velocity*
Ø Air pressure difference measurement*
Ø Airflow direction and visualisation
Ø Microbial airborne and surface contamination*
Ø Temperature measurement
Ø Relative humidity measurement
Ø Recovery testing
Ø “Clean-up phase”
Ø Containment leak testing
* Minimum test requirements for the requalification of cleanrooms

Other characteristics, such as temperature and relative humidity, should be

controlled within ranges that align with product/processing requirements and
support maintenance of defined cleanliness standards (e.g. Grade A or B).

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 2. Measurements for qualification (according to ISO)

2.1 Visual inspection

2.2 Measurement of the pressure cascade between Clean rooms
2.3 Airflow measurement - Volume / velocity
2.4 Installed filter leakage and integrity testing
2.5 Clean room classification
2.6 Measurement of the recovery time (turbulent air only)
2.7 “Clean-up Phase” (Annex 1 only)
2.8 Flow visualization

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 2.1 Visual inspection
The visual inspection should include a review of the filter, wall, floor and
ceiling systems. Irregularities or damage are documented for elimination.
Even the smallest damage to the filter surface indicates a leak.

2.2 Air pressure difference measurement

The measurement of the pressure difference between areas
serves to demonstrate the clean room class distinction! In
addition, permanent monitoring and recording provide evidence
that the clean room has continuously maintained its protective
function.
Specifications for the pressure difference:
ISO 14644-4 → 5 to 20 Pascal per door
Annex 1 → 10 to 15 Pascal per class

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 2.3 Determination of the air speed / number of air changes
By determining the mean air velocity in uni-directional flow homogeneity is
detected, or in turbulent mixed flow, with the air speed measurement, the air
change rate can be determined. The volume flow of air can also be measured
directly. According ISO 14644:
Unidirectional flow:
Distance from the filter outlet area: 150 until 300 mm
Number of measuring points : N =√10×A (ISO)
Turbulent flow, the air change rate is:

ACR = Σ [A * v] / V = Σ V˚/ V
ACR = Air change rate V = Total volume of the room in [m3]
A = Area of the filter in [m] V˚ = Air volume flow for each filter in [m3 / h]
v = Velocity oft the air in [m/s] Σ = Total air volume flow in [m3 / h]

The air exchange rate must be large enough to hold the required clean
room class, but is not specified normative!

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 2.3.1 Determination of the air speed in unidirectional flow
According new Annex 1

The speed of air supplied by unidirectional airflow systems should be

clearly justified in the qualification protocol including the location for air
speed measurement. Air speed should be designed, measured and
maintained to ensure that appropriate unidirectional air movement
provides protection of the product and open components at the working
height (e.g. where high risk operations and product and/or components
are exposed). Unidirectional airflow systems should provide a
homogeneous air speed in a range of 0.36 – 0.54 m/s (guidance value)
at the working position, unless otherwise scientifically justified in the
Contamination Control Strategy. Airflow visualization studies should
correlate with the air speed measurement.

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 2.4 Installed filter leakage test
2.4.1 Specifications

Ø The total air outlet area of a built-in end filter scan coverage
Ø In addition, the filter media and receiving the filter seal must be checked
for leakage limit
Ø The upstream concentration should be selected so that the verification of
the absence of leakage is reliably ensured (minimum)
Ø In addition, the filter medium must not be unnecessarily contaminated by
the test aerosol (maximum)
Ø Particle size of test aerosol: ≤ 1,0 µm

Ø The concentration in the raw air must be demonstrated by measurement

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 2.4.2 Theory for the leak test

The total penetration must not exceed 0.1% for H13

filters and 0.01% for H14 filters.
Assuming that the extent of a leak is small
compared to the probe cross-section, the following
results for the length of time the probe remains
under a leak:
T L = a / vS

The scanning speed vs recommended according to ISO 14644-3: 2020 is <12 cm/s
with the round probe and <5 cm/s with a rectangular probe (15 x 69 mm).

With an expected particle concentration below a nominal leak of 50 particles on the

clean air side per 10 s, the result is a upstream concentration of approx. 3-4 million
particles per cft for an H14 filter.

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 2.4.3 Scanning time for the filter leakage test

Round Probe:
TS = [L * W] / [(Dreal) * vS * 60 s/min]
Rectangular probe :

TS = [(L * W) / Dreal + 2*(L+W)] / [vS * 60 s/min]

TS = Scanning time in [min]
L = Length of the filter in [cm]
W = Width of the filters in [cm]
Dreal = real diameter traverse to the scanning direction in [cm]
vS = scanning speed in [cm/s]

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 2.4.4 Instrumentation for Leakage test

Fan

Air

Aerosol input

Aerosol up stream
HEPA Filter

Aerosol down stream

Dilution system
Faktor 100 - 1'000

Aerosol generator

Particle counter Particle counter

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 2.4.5 Test Aerosol

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 2.4.6 Contamination of the filter during a leak test

Evaporation time

Size [µm] Water DOP DEHS

0,3 160 µs 37 min 4h
1,0 1 ms 8h 57 h
3,0 7 ms 55 h 16 d
10 80 ms 23 d 160 d

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 2.5 Air cleanliness classification according ISO 14644-1 (2015)

ISO - Maximum value of the airborne particle concentration (Particles per cubic meter air)
classification equal or bigger as the particle sizes seen below
number (N) 0,1 µm 0,2 µm 0,3 µm 0,5 µm 1,0 µm 5,0 µm

ISO class 1 10 --- --- --- --- ---

ISO class 2 100 24 10 --- --- ---

ISO class 3 1‘000 237 102 35 --- ---

ISO class 4 10‘000 2‘370 1‘020 352 83 ---

ISO class 5 100‘000 23‘700 10‘200 3‘520 832 ---

ISO class 6 1‘000‘000 237‘000 102‘000 35‘200 8‘320 293

ISO class 7 --- --- --- 352‘000 83‘200 2‘930

ISO class 8 --- --- --- 3‘520‘000 832‘000 29‘300

ISO class 9 --- --- --- 35‘200‘000 8‘320‘000 293‘000

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 2.5.1 Comparison ISO EN DIN 14644–1 (2015) / Annex 1 draft (2020)

Annex 1

Class Particle size at rest in Operation

Particle > 0,5 µm 3.520 3.520

A
Particle > 1,0 µm 832 832
ISO 4.8
Particle > 5,0 µm --* --*

Particle > 0,5 µm 3.520 352.000

B
Particle > 1,0 µm 832
ISO 5 & 7
Particle > 5,0 µm --* 2.900

C Particle > 0,5 µm 352.000 3.520.000

ISO 7 & 8 Particle > 5,0 µm 2.900 29.000

D Particle > 0,5 µm 3.520.000 not define

ISO 8 Particle > 5,0 µm 29.000 not define

* Note in Annex 1: The measurement using a second, larger particle size,

e.g. 1.0 μm according to ISO 14644 can be taken into
account.

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 2.5.2 Calculation of the ISO classification number
Degree of air purity based on the particle concentration, which is used for a clean room or clean area,
expressed in the form of a ISO-class N which the maximum value of the permissible concentration,
expressed in particles per cubic meter of air, for particles equal to or larger than the variables
considered represents.

Formula: Cn = 10N x (0,1/D)2,08

Where:
The maximum value of the permissible concentration (in particles per cubic meter of air) of airborne particles, which of or
larger than the given particle size are equal.
Cn is rounded to the nearest whole number, with no more than three valid numbers are used
N is the ISO classification number, which must not exceed a value of nine or less than 1. Intermediate ISO
classification numbers may be specified, with 0.1 permissible as the smallest increment of N;
D the given particle size expressed in microns;
0.1 a constant size expressed in microns.

Example:
For Class A GMP follows: D = 5,0 µm and Cn = 20 particles N = log [20 / (0.1 / 5) 2.08] = 4.8
For Class A and B GMP follows: D = 0,5 µm and Cn = 3’520 particles N = log [3’520 / (0.1 / 0,5) 2.08] = 5
For Class A and B GMP follows: D = 1,0 µm and Cn = 832 particles N = log [832 / (0.1 / 1,0) 2.08] = 5
For Class C GMP follows: D = 5,0 µm and Cn = 2’900 particles N = log [2’900 / (0.1 / 5) 2.08] = 7
For Class D GMP follows: D = 5,0 µm and Cn = 29’000 particles N = log [29’000 / (0.1 / 5) 2.08] = 8

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 2.5.3 Minimum sample volume
At each measuring position a minimum sample volume is to be taken, with which at least 20
particles of the largest regarded particle size are measured. For the ISO classes 4.8 to 8
and a regarded particle size ≥5,0 µm is the minimum measuring time:

Formula: V = [20 / cn,m] x1000

Particle Limit Particle Limit Particle Limit Measuring time

ISO – Sample volume
for 1 CFM
Class-No. for ≥ 0,5 µm for ≥ 1,0 µm for ≥5,0 µm in Liter
Particle counter
4.8 20 1‘000 36 - 7**
5 3‘520 832 29 5,7 - 24,0 - 690 1 - 1 - 25 - 5**
6 35‘200 293 68,3 3
7 352‘000 2‘930 6,80 1
8 3‘520‘000 29‘300 0,68 1

** With application of the successive sampling procedure

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 2.5.4 Numbers of Measuring points:

Number Number Number

Area [m2] Area [m2] Area [m2]
MP MP MP
1 1 36 9 116 18
2 1 52 10 148 19
4 2 56 11 156 20

6 3 64 12 192 21

8 4 68 13 232 22

10 5 72 14 276 23
24 6 76 15 352 24

28 7 104 16 436 25
32 8 108 17 636 26

For rooms larger than 1‘000 m2 : NL = 27 x A / 1‘000

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 2.5.5 Arrangement of sampling locations

Ø With a risk analysis the measuring points have to be defined

Ø The minimum number of sampling locations, NL, is to be determined from the table
Ø First, the risk points are determined
Ø Then the entire clean room or the clean area must be subdivided into equal NL sections,
Ø Within each section, select a sampling site which is considered to be representative of
the characteristics of the section and
Ø At each sampling point, the Isokinetic probe should be placed at the level of the working
activity or at another specified point (e.g. 1,2 m over flour).
Ø Additional sampling locations may be selected for locations that are considered critical.
Their number and location must also be agreed and determined.
Ø To facilitate the division into equal sections, additional sections and associated sampling
locations may be included.
Ø For clean rooms or clean areas, sampling locations may not be representative if they
are located directly below the uni-directional flow.

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 2.5.6 Evaluation of the cleanroom class

Each point of measurement is considered for itself.

The particle concentration (given in particle number per cubic meter) in the individual
sample volume at each sampling location shall not exceed the concentration limits for
the variables considered in the table after completion of the tests. If several individual
sample volumes are taken at a sampling site, the concentration shall be averaged
and the mean concentration shall not exceed the concentration limits indicated in the
table.

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 Example:
Position Probe 1 Probe 2 Probe 3 average / cft average / m3 limit Pass / fail

1 47 57 52 1‘836 3‘520 pass

2 12 12 424 3‘520 pass
3 162 78 32 91 3‘201 3‘520 pass
4 148 74 132 118 4‘165 3‘520 fail
5 1 0 0,5 18 3‘520 pass
6 19 22 17 19 682 3‘520 pass
7 5 15 3 8 271 3‘520 pass
8 38 21 30 1‘041 3‘520 pass
9 54 159 78 97 3‘420 3‘520 pass
10 48 62 53 54 1‘918 3‘520 pass

At sample location 4, the mean sample volume concentration of 4'165 does not meet the criteria of the
highest particle count of 3'520 of ISO class 5. At sampling site 3 and sampling location 9, one of the
concentrations of the individual particle counts does not hold the limit set in Table 1. However, neither the
mean particle concentration for sampling site 3 nor for sampling site 9 violates the limit set in Table 1.
Since the air purity is not met by the particle concentration at sample location 4, the clean room does not
meet the required classification.

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 2.6 Recovery time

Ø Direct method (100-to-1 Recovery time)

The 100: 1 - Recovery time is defined as the time, which is
necessary, in order to reduce a particle concentration to 1% of their
initial value. if this is not possible, the indirect method can be used.
Ø Measuring height
According the cleanroom class (1.2 m above ground)

Ø Selection of the measuring location

The measuring location should not be selected directly under an air
outlet. One selects a range in which the particle concentration in the
clean room class determination is largest (highest particle
concentration -> worst dilution -> longest recovery time.)

Ø Considered particle size

The smallest particle size channel of the particle counter

Important: There is no normative Limit for the recovery time!

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 Particle measurement to observe the recovery time
Pos. R Date 12.03.2008
TIME >0.30μm >0.50μm >1.00μm >5.00μm >10.00μm %FLOW
09:10:55 411 114 26 4 4 1.00
09:11:55 198428 72390 8297 9 1 0.99
09:12:55 100117 22121 1519 5 1 0.99
09:13:55 69215 15127 1016 4 0 1.00
09:14:55 46655 10192 711 4 0 0.99
09:15:55 32069 6901 442 2 0 1.00
09:16:55 22102 4826 323 1 0 0.99
09:17:55 12148 2667 190 1 0 0.99
09:18:55 3777 847 61 0 1 0.99
09:19:55 1786 531 47 0 1 0.99

Max 198428 72390

1% of the maximum value 1984 723
Maximum value reached at 09:11:55 09:11:55
1st value <1% of the
maximum value 1786 531
Value reached at 09:19:55 09:19:55
Recovery time 00:08:00 00:08:00

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 2.7 „Clean-up“ Phase
Immediate measurement of the clean-up phase
The particle limits in the table of the cleanroom classification should be reached
after a short clean-up phase of 15-20 minutes (guideline) in an unmanned state
after work is completed.
At one or more representative measuring points in the room, particle
measurements are continuously carried out to obtain a decay curve for the
transition from the operating state "in operation" to the operating state "at rest".
100000

10000 Limit "At rest"

Limit "In operation"
Test 1
Test 2
1000
Test 3

100
0:00 0:04 0:08 0:12 0:16 0:20 0:24

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 2.8 Flow visualization

Flow visualizations or smoke studies must be performed to demonstrate that:

ØThe required flow direction is maintained (for example in pressure cascades).
ØIn the uni-directional flow to detect the absence of air from the surrounding area
ØIn turbulent mixed flow to detect the absence of so-called "flow-free" areas
Annex 1:
Airflow patterns within cleanrooms and zones should be visualised to demonstrate that
there is no ingress from lower grade to higher grade areas and that air does not travel
from less clean areas (such as the floor) or over operators or equipment that may
transfer contaminant to the higher grade areas. Where air movement is shown to be a
risk to the clean area or critical zone, corrective actions, such as design improvement,
should be implemented. Airflow pattern studies should be performed both at rest and in
operation (e.g. simulating operator interventions). Video recordings of the airflow
patterns should be retained. The outcome of the air visualisation studies should be
considered when establishing the facility's environmental monitoring program.

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 3. Overview of the periodic tests
ISO 14644* / VDI 2083-2:2015 EG Guideline
ISO 4.8 ISO 5 ISO 6 - 9 A B C-D

Visual inspection At every periodic test

Tightness cleanroom At the first qualification

Pressure room 6M 6M 12 M 6M 6M 12 M
Air changes n. a. 6M 12 M n. a. 6M 12 M
Pressure drop filter 12 M 12 M 12 M 12 M 12 M 12 M

Filter leakage test 6M 6M 12 M 6M 6M 12 M

Classification 6M 6M 12 M 6M 6M 12 M
Recovery time At the first qualification and changes ---
Clean-up-phase --- Ongoing, interval defines the operator

visualization At the first qualification and changes

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 4. Documentation
Structure of the measurement protocols according to ISO EN DIN 14644-1:
The results of every re-qualification or test of a system to demonstrate continuous
compliance must be recorded in a comprehensive report and submitted together with a
declaration of compliance or non-compliance with the specified tests.
The following data must be included in the measurement protocol:
Ø Name and address of responsible testing group and date on which the test was carried
out,
Ø The number and date of issue of the International Standard used
Ø Clear identification and location of the tested clean room or clean area (including a
reference to the adjacent areas, if necessary) as well as the specified identification of
the coordinates of all sampling locations (a schematic drawing can be helpful),
Ø The specified characterization of the criteria for the clean room or clean area, including
the ISO classification, the relevant operating state (s) and the particle size (s) under
consideration,
Ø Details of the test procedure used with all special features that are related to the test or
deviations from the test procedure and identification of the test device and its valid
calibration certificate and
Ø The test results, including particle concentration data, for all sampling locations

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