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org/aber Advances in Biomedical Engineering Research (ABER) Volume 2, 2014

18
Strategies for the assessment of Disinfection
and Cleaning on Biopharmaceutical
Cleanroom
Authors: *Biunayki R. Daz
1,a
; Caridad A Gasmuri
1
; Noelia Baltrell Mena
1
; Amaury Hierrezuelo
1
; Lzaro
Estenoz
2
; Denis Alvarez
2
; Yadira Mora
3
and Alejandro B. Iznaga
(1)
Microbiology control Group of process control department,
(2)
Bacteria Production Group,
(3)
Quality Assurance
Direction
Center for Genetic Engineering and Biotechnology. Ave 31 be/158 and 190, P.O.Box 6162, Havana 10600, Cuba
a
biunayki.reyes@cigb.edu.cu

Received Dec 14, 2013; Revised Feb 18, 2014; Accepted Mar 15, 2014; Published Jul 9, 2014
2014 Science and Engineering Publishing Company


Abstract
Cleaning and disinfection assessment is an integral part of
current good manufacturing practices in any pharmaceutical
industry. Nowadays, Interferonand several other
pharmacologically potent biopharmaceuticals are
manufactured in same production area. Carefully designed
cleaning and its assessment can ensure that residues of
disinfectant used in the sanitization will not carry over and
cross contaminate the subsequent product as well as any
residual. The qualification of procedure was carried out on a
screening test in order to measure effectiveness of the
disinfectants, chosen according to their principle of action.
This article intended to address special considerations and
issues pertaining to assessment of cleaning and sanitization
procedures for cleaning areas to the manufacture of
biopharmaceutical products, and biological drugs. Its also
intended to cover the control of potential microbial
contaminants associated with the clean areas.
Keywords:
Cleaning Assessment; Biopharmaceutical Product; Disinfectant;
Sanitization; Good Manufacturing Practices; Microbial
Contaminants
Ar t i c l e Ac r onym Li st i ng
C-GMP: current good manufacturing practices; FDA:
food and drug administration; DNA: deoxyribonucleic
acid; Mabs: monoclonal antibodies; SOP: standard
operating procedure; CFU colony forming units
I nt r oduc t i on
The current good manufacturing practices (c-GMP's)
regulates the assessment of the cleaning processes in
the pharmaceutical industry (21 CFR 211.67, 2006) and
(21 CFR 211.60, 2006). The Food and Drug
Administration (FDA) enforced those cleaning
processes and the agency published a guide where
they specified that no detergent should remain after
the cleaning process (FDA, 1993). It is common to find
reports on cleaning assessment of drug residues
(Lakka, 2014, Katona, 2000, Nozal, 2002 and
Klinkerberg, 2003). However, reports on cleaning
assessment of detergents used for the cleaning process
are limited (Yang, 2005) and (Zayas, 2006). The
detection of traces of detergents is a difficult task in
cleaning assessment programs.
Pharmaceutical, bio-medical device and even food
preparation industries are concerned about the
cleanliness - physical, chemical and especially
biological - of their products. These industries have
materials that need regular careful cleaning, and some
have requirements for validating such cleaning,
demonstrating that required levels of cleanliness has
been met. They want to prove the efficacy of their
cleaning methods. For example, "Cleaning and
assessment of cleaning are among the most critical
issues facing producers of recombinant DNA protein
products, monoclonal antibodies (MAbs), and
oligonucleotide therapeutics," according to Adner and
Sofer, 1994. As noted below, the Food and Drug
Administration (FDA) has taken assessment of
cleaning very seriously. It is widely recognized that
contamination control is crucial to these industries.
Here we deal with the cleaning of surfaces.
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19
The production and cleaning operations involved in
the production area should follow strict good
manufacturing practices. Among these are cleaning
assessments, which is critical for patients safety and
health involved in the production. Moreover, the
cleaning assessment is integral part of quality
assurance that embodies all the necessary steps to
guarantee the quality of medications be inside the
adopted standards, be safe and effective for
therapeutic application (Moser, 2000).
During the cleaning assessment, following factors
should be taken into consideration: construction
material, surfaces of the facilities and equipment that
offers greater risk of contamination. It is important to
standardize cleaning procedures and cleaning
material, verification of residues chemical products
and post-cleaning microbial load. Other factors as the,
sampling procedure should also be considered
[Canada Health Products, 2003].
The acceptable limit for residue in the equipments is
not established in the current regulations. However,
for the cleaning areas and its classification the Food
and Drug Administration (FDA), ISO 14644 and
USP<1116> classified the clean areas according to the
classification approaches of acceptance criteria
microbiologic (Sartain, 2004)
The objective of this work on cleaning assessment was
to prove, through validated analytical method, that the
cleaning procedure was efficient in removing product
residues and excipients, degradation products,
cleaning substance and other possible contaminants.
This cross contamination risk in production area can
be reduced substantially.
Ex per i ment al
The flowchart in Figure 1 graphically shows the
different aspects that should be considered when
developing a cleaning assessment program.
Understanding each aspect of the process, the
relationships among these actions, and the sequence in
which they should take place would make the
development of a cleaning assessment program
successful.
The figure shows the fourth is most important stepin
the assessment based on the validation master plan, as
well as aspects such as operating procedures,
sampling points and their methods, types of surfaces
to be sampled; the very important step is the
description products that are manufactured in clean
areas, for better data compression meet acceptance
criteria according to regulations established internally
and on test methods, all these aspects are important to
consider and should not be missed in the preparation
of the protocol which is one of the main topics
discussed in this article

FIGURE 1 FLOW CHART OF CLEANING AND DISINFECTION
ASSESSMENT
Materials
One objective of surface sampling was to determine
the efficiency of routine cleaning procedures in
removing contamination. Sampling was done before
and after sanitization. The medium in the plates
contains neutralizing agents, which inactivate residual
disinfectants on the surface to be tested, allowing
comparative results before and after cleaning
Surface Monitoring
ISO 14644, Fed Std- 209E, USP <1116> Surfaces
monitoring sanitized with 70% ethanol are conducted
using prepared contact plates filled with Tryptic soy
agar (TSS) (casein-peptone soymeal peptone agar)
(MERCK) and the surface sanitized with disinfectant
(Bactylisine and Surfalyse) are conducted using
prepared contact plates with Tryptic soy agar with
polisorbate 80 and lecithin (TLC) (RODAC plates, 65 x
15 mm). The exposed plates are incubated to promote
growth, the microorganisms are counted and results
are reported as the number of CFU (colony forming
units) per area sampled.
Flat agar surface is above the edges of the dish (so can
press it on flat surfaces) and a grid, allowing counting
of cfu per cm.
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Sampling
The sample surfaces of the areas and equipment were
carried out before the cleaning and 1 hour after
concluding this operation for the method of media
plate according to the corresponding SOP, in order to
guarantee an appropriate recovery of the
microorganisms present in the sampled surfaces. The
annotations would be carried out in assessment
register (figure 2). For each selected point, we carried
out sampling 3 times operations of cleaning and serial
sanitation. Once taken the sample, they were taken
until the Quality Microbiology Laboratory.
1) Clean Rooms Monitored
To carry out this work, the production area used was a
multi-product factory for purification of Active
Pharmaceutical Ingredient (API) of a group of
recombinant products with a common host cell,
Escherichia coli and similar production processes. This
area had walls and floors of polyvinyl chloride (PVC),
areas B grade laminar flow ceiling and laminar flow
grade A
Sampling points at each location of the factory
Local
Clean areas
class
Sampling surface
sampling
point code
Lock input Gray /
White
Grade D Wall surface Sp1
Local Support Grade C Wall surface Sp2
staff entry lock Grade D
Wall surface Sp3
Floor surface S1
clean corridor Grade D
Wall surface Sp4
Floor surface S2
Support local Grade C
Wall surface Sp5
Floor surface S3
surface of furniture PC1
Preparation of
solutions
Grade C
table surface PC2
surface of furniture PC3
surface equipment PC4
Floor surface S4
Floor surface S5
Local Extraction Grade C
Wall surface Sp6
Wall surface Sp7
Wall surface Sp8
surface equipment PC5
Floor surface S6
surface of furniture PC6
horizontal
laminar flow
cabinet
Grade A in
Grade B
curtain surface PC7
curtain surface PC8
Table surface PC9
surface equipment PC10
Floor surface S7
Purification local Grade B
Wall surface Sp9
surface of furniture PC11
Wall surface Sp10
Ceiling laminar
flow
Grade B in
Grade C
curtain surface PC12
curtain surface PC13
Leyend: Sp (Wall surface); S (floor surface) and PC
(other surfaces)
The plates were incubated and invested by a period
(According to USP chapter <1116>; incubation
temperatures should be in the 22.5 2.5 C and 32.5
2.5 C ranges with an incubation time of 72 and 48
hours respectively) (USP 1116, 2012); having care with
their manipulation was of soft way and once they are
incubated, don't move again until concluded the
period of incubation. The count carried out
enumerating the UFC present in the plate in
accountant colonies, this procedure of incubation was
carried out according to SOP respectively.

FIGURE 2 REGISTER MODEL DESIGNED TO REGISTRATION OF
MICROBIOLOGY SAMPLING ASSESSMENT FOR EACH
SELECTED POINT IN THE CLEAN ROOM
Personnel
It is difficult to validate a manual cleaning procedure,
i.e. an inherently variable/cleaning procedure.
Therefore, operators carrying out manual cleaning
procedures should be adequately trained, monitored,
and periodically assessed. The success of a disinfectant
program is directly related to the quality of training
provided to the personnel charged with executing the
program and the degree to which they comply with
the disinfectant protocol. Most disinfectant program
failures occur either because the wrong chemistry was
selected or because the right chemistry is not applied
properly. Training of the personnel included basic
microbiology and basic chemistry related to
disinfectant preparation and application including
safety, aseptic techniques, and a job-related standard
operating procedure based upon proper testing and
assessment (Wenzel, 2002).
Disinfectants
Disinfectants were selected on the basis of
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21
performance against common environmental
contaminants and more than one product must be
included in the disinfectant program in order to
achieve broad-spectrum performance. The program
included routine disinfectants, sporicides, and residue
reducing agents (Vellutato, 2006).
1) Solution Disinfectant
Alcohol (Ethanol 70%: antiseptic and antiviral agent).
Quaternary ammonium compounds (Neosteril 2%:
Germicide- fungicide). Bactilysine Germicide
Aldehidos (Surfalyse 0.25%: Antiviral, sporicides and
fungicide).
One of the main limitations of the study is the
availability of suitable disinfectants, as there are
different factors that affect the action of these
disinfectants, among which we can mention:
Time performance and product concentration: usually
by increasing the contact time increases the fatality
rate. The contact time is one of the critical factors to
ensure disinfection. Usually been experimentally
established a time of 5 minutes in which time can be
determined by lethality bacterial sanitizers
Surface action: The choice of materials and processing
was performed on the surfaces in order to retain the
smallest number of bacteria after cleaning and
disinfection was crucial in order to ensure the hygiene
of the surfaces and thus prevent cross-contamination
Effectiveness of Sanitizers
1) Inoculum Preparation
The microorganisms were grown on plates containing
sporulation medium (MnCl2 4H2O 10 mg / mL) for 5
days at 30 to 35 C
The procedure was to inoculate of a coupon simulate
surfaces which were to disinfect clean room
materials such as (stainless steel, glass, polycarbonate,
PVC and polypropylene) with the target organism,
followed by exposure to the disinfectant solution.
The inoculums level was sufficient to account for the
percent recovery of the chosen method (10
5
to 10
6
cfu/cm
2
) and immediately was dried at room
temperature (19-21 C) in aseptic condition. Then was
allowed act for 10 minutes the sanitizing subsequently
all surfaces were rinsed with a solution composed of
0.1% peptone , 0.05% Tween 80 and 0.07% lecithin to
neutralize the effects of sanitizing , filtered through a
membrane of nitrate or acetate cellulose 0.45 um , the
membrane was placed in a plate containing tryptic
soy agar with neutralizer , then incubated 72 hours to
5 days, at a temperature of 30 to 35 c , bacteria and
yeast for 20 to 25 c and up to 7 days, the filamentous
fungi of 20 to 25 c CFU counted present therein as
recommendations related to the regulation.
It should be noted that in terms of wiping, some of the
apparent microorganism reduction may be due to the
mechanical action of the application techniques rather
than the activity of the disinfectant. Therefore, the
proper control (wiping or immersion with a water
solution) was included in this type of evaluation.
The sanitizers were regarded as effective against
vegetative bacterial cells (bactericide) if attaining, at
least, a 3-log c.f.u. reduction compared to that of
control microorganisms (P. aeruginosa, S. aureus,
Staphylococcus spp. and micrococci isolated from the
manufacturing facility environment). Similarly, they
were regarded as effective against bacterial spores
(sporicidal) and fungi spores (fungicide) if attaining at
least a 2-log reduction vs controls (B. subtilis and
sporulated grampositive environmental, A. niger and
filamentous fungi from the manufacturing
environment, respectively). These acceptance criteria
were taken as explained by the USP informational
chapter <1072>.
Documentation
Assessment studies were the foundation of the
regulatory submission. They required a large amount
of work in a short period of time. For a particularly
complicated study, several months may be required
for proper assembly, review, and approval of the
assessment package. Allow enough time to perform
the studies, write the reports, and ensure that the
documentation was in order before the results went
into the submission (Feuerhelm, 2001).
Protocol
Before we were beginning the assessment, we wrote
and approved a protocol. FDA defined a protocol as a
written plan stating how assessment will be
conducted, including test parameters, product
characteristics, production equipment, and decision
points on what constituted acceptable test results
(EC, 2008 and Annex15 EU, 2001). Protocols contained
five sections: the purpose and scope, an introduction, a
process description, materials and methods, and the
acceptance criteria (Figure 3) [PIC/S, 2007 and
CECMED, 2012].
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FIGURE 3 PRINCIPAL STEP DISINFECTION AND CLEANING
ASSESSMENT PROTOCOL
1) Purpose And Scope
Begin the protocol by stating the purpose and scope of
the assessment. For an in-process hold time study, you
might write, The purpose of this study was to
demonstrate that the quality of the product was
maintained during the process which held steps used
in the manufacturing process.
2) Introduction
Next, the introduction addressed responsibilities,
criteria for assessment (such as contaminant levels). It
is important to address who will be responsible and
for each task. Being specific ensures that everyone
knows with whom each responsibility lays. That will
keep the study and documentation flowing smoothly
and should prevent bottlenecks.
3) Process
Following the introduction, describe the process, and
address which parts of it will be validated.
4) The Materials And Methods
Section describes how a study will be performed.
Sampling, equipment, materials, and assays appear in
this section. When addressing sampling, be specific.
List the sample points, a mistake in sampling can be
costly and require the study to be repeated.
5) Acceptance Criteria
Finally, every protocol should have an acceptance
criteria section. These criteria must be met if the
assessment study is to be considered successful. The
criteria should be descriptive and clearly defined. A
clear and specific protocol would make writing the
summary report easier and help assure success of the
assessment study.
Resul t s and Di sc ussi on
Areas hardest to clean and reasonably accessible were
evaluated by direct sampling method, leading to
establishing a level of contamination or residue per
given surface area. The suitability of the material to be
used for sampling and of the sampling medium was
determined. The ability to recover a sample accurately
may be affected by the choice of sampling material.
For instance, its important to assure that the sampling
medium and solvent (used for extraction from the
medium) were satisfactory and can be readily used.
The frequency of cleaning and disinfection will
depend on the classification of the cleanroom and the
operation within it. This will also affect the choice of
application method to achieve optimum results. We
classified our clean areas following the criteria of the
World Health Organization (WHO). To test
antimicrobial effectiveness, we will culture the chosen
isolates to an enumeration nearing 1.0 x 10
4
, though
some may argue a higher enumeration is needed. The
justification for the stated enumeration is that when
RODAC samples are taken in a Class 100 (Grade A,
ISO 5) area, most firms set their action levels at 1 to 2
colony forming units (CFUs). In Class 10,000 (Grade C,
ISO 7) areas, the action levels are normally 5 to 10
CFUs and in Class 100,000 (Grade D, ISO 8) areas,
most firms have set the action levels at 25 to 50 CFUs.
Methodology
The USP 1072 document provides input on
disinfectant assessment, an area of particular concern
to regulatory authorities such as the Food and Drug
Administration or the European Medicines Agency.
Disinfectant assessment includes not only in vitro
studies demonstrating a disinfectants performance
against specific organisms under controlled conditions
but also an environmental assessment of how the
disinfectant is performing under actual use conditions
[USP, 2012]
Procedure qualification was carried out screening
disinfectants for their efficacy at various
concentrations and contact times against a wide range
of standard test organisms and environmental isolates
Initially in order to measure the disinfectants
effectivenesswhich was chosen according to their
principle of action, in materials present in area and

Introduction

Purpose

Responsibili
ties

Develop
Assessment
Acceptance
Approaches
Reassessme
nts /
Changes
Control

A As ss se es ss sm me en nt t
R Re ep po or rt t
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23
later on the acting of the cleaning was qualified for
which they were carried out races with the plant in
operation ("in process") and sampled significant points
inside the premises of work (Table 1 A and B), as one
could appreciate, the disinfectants completed the
approach of acceptance with the screening,
demonstrating their action germicide.
TABLE 1 A SCREENING TEST FOR EFFICACY METHOD DISINFECTANT SURFALYSE
microorganism
Acceptance criteria PVC Floor Wall PVC Curtain
Logarithmic reduction start end Log red start end Log red start end Log red
Pseudomonas aeruginosa ATCC 9027 3 3,2x10
7
< 10
2
>5 3,2x10
4
<10
2
>5 3,2x10
7
<10
2
>5
Staphylococcus aureus ATCC 6538 3 5,0x10
7
<10
2
>5 5,0x10
4
<10
2
>5 5,0x10
7
<10
2
>5
Bacillus subtilis ATCC 6633 2 7,0x10
5
4,2x10
3
2,2 7,0x10
5
3,0x10
3
2,4 7,0x10
5
4,1x10
3
2,2
Candida albicans
ATCC 10231
3 2,8x10
5
<10
2
>3 2,8x10
5
<10
2
>3 2,8x10
5
<10
2
>3
Aspergillus brasiliensis ATCC 16404 2 1,7x10
5
2,8x10
2
2,8 1,7x10
5
2,0x10
2
2,9 1,7x10
5
<10
2
>3
environmental Bacillus 2 6.0x10
5
2,7x10
2
3,3 6,0x10
5
2,0x10
4
1,5 6,0x10
5
7,5x10
2
2,9
environmental Micrococcus 3 3,8x10
6
<10
2
>4 3,8x10
6
<10
2
>4 3,8x10
6
<10
2
>4
environmental
Staphylococcus
3 4,6x10
6
<10
2
>4 4,9x10
6
<10
2
>4 4,9x10
6
<10
2
>4
environmental
Filamentous fungi
2 3,4x10
5
2,0x10
2
3,2 3,4x10
5
3,0x10
2
3,1 3,4x10
5
3,2x10
2
3,0
TABLE 1 B SCREENING TEST FOR EFFICACY METHOD DISINFECTANT BACTYLISINE
microorganism
Acceptance
criteria
PVC Floor Wall PVC Curtain
Logarithmic
reduction
start end
Log
red
start end Log red start end Log red
Pseudomonas aeruginosa ATCC
9027
3 3,2x10
7
<10
2
>5 3,2x10
7
<10
2
>5 3,2x10
7
<10
2
>5
Staphylococcus aureus
ATCC 6538
3 5,0x10
7
<10
2
>5 5,0x10
7
<10
2
>5 5,0x10
7
<10
2
>5
Bacillus subtilis ATCC 6633 2 7,0x10
5
7,0x10
2
3 7,0x10
5
1,0x10
3
2,8 7,0x10
5
2,0x10
3
2,5
Candida albicans
ATCC 10231
3 2,8x10
5
<10
2
>3 2,8x10
5
<10
2
>3 2,8x10
5
<10
2
>3
Aspergillus brasiliensis ATCC
16404
2 1,7x10
5
<10
2
>3 1,7x10
5
<10
2
>3 1,7x10
5
<10
2
>3
environmental Bacillus 2 6,0x10
5
1,0x10
3
2,8 6,0x10
5
2,0x10
3
2,5 6,0x10
5
<10
2
>3
environmental Micrococcus 3 3,8x10
6
<10
2
>4 3,8x10
6
<10
2
>4 3,8x10
6
<10
6
>4
environmental
Staphylococcus
3 4,9x10
6
<10
2
>4 4,9x10
6
<10
2
>4 4,9x10
6
<10
2
>4
environmental
Filamentous fungi
2 3,4x10
5
<10
2
>3 3,4x10
5
<10
2
>3 3,4x10
5
<10
2
>3

This is considered necessary because critical process
steps like disinfection of aseptic processing areas, as
required by GMP regulations, need to be validated.
Qualification Process
The removal of particulates, microbes and possibly
existent residues from surfaces is characterized as
cleaning, and it requires that a non-destructive
mechanical action be applied to loosen and remove
contaminants from the area. Procedurally,
contaminants and residues are loosened and rinsed
to the floor. Subsequently, the dirtied solution on the
floor is collected and removed from the area. By
lessening the level of particulates, microbes and
residues on the surface, disinfection efforts become
simpler. First, there are fewer organisms to destroy
because most have already been removed from the
area. And secondly, as bioburden and residue levels
decreased, the possible obstructions blocking the
chemical agent from contacting the organism are
minimized. In short, cleaning prepares a surface for
disinfection. Initially, before the cleaning, we carried
out a sample for contact plate methods of each one of
points defined on protocol of assessment to Plants,
that they were selected of such way that includes
walls, floors, teams and other surfaces
Keeping in mind the written by (Vellutato 2006)
We have proven in a controlled laboratory test that a
validated sanitizer, disinfectant or sporicide can
destroy a known enumeration of microorganisms on
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24
a carrier surface, but what happens when we
combine the validated sanitizer, disinfectant or
sporicide with our cleaning SOPs and our personnel?
Will they all work together to attain success? Having
assessment data for the antimicrobial effectiveness of
the agents does not guarantee the area will be
cleaned and disinfected; therefore, most firms
conduct an in situ or field study (Feuerhelm, 2001).
Well, I would dare to say that here this answer to this
questions.
Were defined three samples hours and following a
chronological process:
Zero hours (sampling before cleaning and
disinfection beginning)
Hour 1, (after carrying out the cleaning and
disinfection of the areas)
Hour 8, (holding time; to avoid microbial
proliferation It was expected to demonstrate they
maintained acceptable microbial quality between the
time the surfaces areas and equipment were cleaned
and the time were used)
The total established points for the interest area,
were from 61 points corresponding to the most
critical zones, according to the step of the process to
carry out.
As we can see on (Figure 4), graphically could
observe the behavior of results in the three days of
carrying out qualification for the areas classified like
GRADE C.

FIGURE 4 BEHAVIOR OF DISINFECTANT PROGRAM RESULTS IN THE THREE DAYS OF CARRYING OUT QUALIFICATION FOR THE
AREAS CLASSIFIED GRADE C
We considered that the cleaning and disinfection
carried out with Bactylisine 0.25% and Ethanol 70%;
Surfalyse and Ethanol 70 %, was effective; since all the
points completed the acceptance approach established
to the hour of carrying out the cleaning and sanitation.
Upon analyzing the Holding time of the cleaning eight
hours later, one single point was observed that don't
complete the approach of acceptance of 25 [ufc]/ cm
2
,
in order to deepen in the approaches, was carried out
the analysis of specific points that they didn't fulfill the
approach of acceptance in the three days of carrying
out samples, valuing the type of point and the activity
that around them were carried out when the area was
in operation.
For clean room classified GRADE D, all the points
fulfilled the acceptance approach the three days, in the
three sampling hours. However for case of disinfectant
bactylisine in clean room GRADE C (Figure 4), alone
Disinfectant Surfalyse zero hour
clean room Grade C
0
5
10
15
20
25
30
P
C
1
0
P
C
1
5
P
C
2
0
P
C
2
5
P
C
3
0
P
C
9
S
1
3
S
p
1
0
S
p
1
5
sampling points
u
f
c
/
c
m
2
day 1
day 2
day 3
approach
Disinfectant Surfalyse Hour 1
clean room Grade C
0
5
10
15
20
25
30
P
C
1
0
P
C
1
5
P
C
2
0
P
C
2
5
P
C
3
0
P
C
9
S
1
3
S
p
1
0
S
p
1
5
sampling points
u
f
c
/
c
m
2
approach
acceptance
day 1
day 2
day 3
Disinfectant Bacylisine zero hour
clean room Grade C
0
5
10
15
20
25
30
P
C
1
0
P
C
1
4
P
C
1
8
P
C
2
2
P
C
2
6
P
C
3
0
P
C
8
S
1
1
S
2
S
p
1
1
S
p
1
5
S
p
9
sampling points
u
f
c
/
c
m
2
day1
day2
day3
Desinfectant Bactylisine hour 1
clean room Grade C
0
5
10
15
20
25
30
P
C
1
0
P
C
1
4
P
C
1
8
P
C
2
2
P
C
2
6
P
C
3
0
P
C
8
S
1
1
S
2
S
p
1
1
S
p
1
5
S
p
9
sampling points
u
f
c
/
c
m
2
approach
acceptance
day1
day2
day3
Advances in Biomedical Engineering Research (ABER) Volume 2, 2014 www.seipub.org/aber
25
one point didn't fulfill the approach of acceptance to
the initial hour and to the eight hours. It happened the
same when we evaluated the disinfectant Surfalyse.
TABLE 2 RELATIONSHIP OF POINTS THAT DIDN'T FULFILL THE ACCEPTANCE APPROACH IN ZERO HOUR, BEFORE CARRYING OUT CLEANING WITH
DISINFECTANT BACTYLISINE AND ETHANOL FOR CLEAN ROOM GRADE C
Point Description Type of point
Day Time NC
Activity associate
1 2 3
S10 Floor, LFT center point located in the floor X Purification
S10 Floor, LFT center point located in the floor x Purification
TABLE 3 RELATIONSHIP OF POINTS THAT DIDN'T FULFILL THE ACCEPTANCE APPROACH IN ZERO HOUR, BEFORE CARRYING OUT CLEANING WITH
DISINFECTANT SURFALYSE AND ETHANOL FOR CLEAN ROOM GRADE D
Point Description Type of point
Day Time NC
Activity associate
1 2 3
S9 Floor, LFT center point located in the floor x Purification
S1 Floor, area center point located in the floor x Purification

We made the analysis of points types using statistics
not parametric considering the discreet character and
not associated with the obtained results. Friedman
test using STATGRAPHICS CENTURION XV
program. The test of Friedman evaluates the null
hypothesis that the medium inside each one of the 3
columns (corresponding to the sampling days it is the
same. Since the P-valor is minor that 0.05, a statistical
difference between the medium with a level of the
95.0% of trust.
In order to determine who medium they were
significantly different of another, we selected the
Chart of Box and Mustaches (Figure 5 and Figure 6)
Friedman test for (S) Point clean room ISO 7 using
disinfectant Bactylisine
Sample size average range
S (zero hour) 27 2.5
S (1 hour) 27 1.57407
S (8 hour) 27 1.92593
Statistics = 13.6989 P-value = 0.00106003
Disinfectant BactylisineceanroomISO 7
answer
zerohour)
S(hour 1)
S(hour 8)
0 5 10 15 20 25 30

FIGURE 5 CHART BOX AND MUSTACHES FOR CLEAN ROOM
ISO 7 (BACTYLISINE DISINFECTANT)
Friedman test for (S) Point clean room ISO 7 using
disinfectant Surfalyse
Sample size average range
S (zero hour) 27 2.59259
S (1 hour) 27 1.83333
S (8 hour) 27 1.57407
Statistics = 16.34 P-Value = 0.000283018
Disinfectant Surfalyse cleanroomISO7
answer
zero hour)
S (hour 1)
S (hour 8)
0 25 50

FIGURE 6 CHART BOX AND MUSTACHES FOR CLEAN ROOM
ISO 7 (SURFALYSE DISINFECTANT)
According to the protocol used to validate the
cleaning, we carried out the corresponding
investigation to no conformity which happened
during the same, and we evaluated the sampling
points that were from acceptance approach. The
procedures of cleaning and sanitation would be
evaluated periodically in order to confirm that they
continued being valid every 2 years.
When significant changes concerning the validated
state have not taken place, this necessity of
reassessment would be covered with a statistical
historical revision that demonstrated the processes of
www.seipub.org/aber Advances in Biomedical Engineering Research (ABER) Volume 2, 2014
26
cleaning completed the demands and approaches of
pre-established acceptance in the analyzed period.
Conc l usi ons
Developing, validating and assuring the
implementation of an overall system for cleaning and
disinfection are critical. Without a system-based
approach, the efforts will be thwarted by problematic
situations. The control of contamination, the review of
environmental monitoring data, the conducting and
review of assessment data, the appropriate
application, and continued supervision and training
all combine to ensure success.
The assessment activities of cleaning cover the
identification of the microorganism, the selection of
sample method, establish the approach of acceptance
for residual, assessment of the method, studies of
recovery.
The success obtained of assessment program for
cleaning to been due to a good preparation and
implementing appropriate the tools of cleaning and
disinfection assessment.
The procedures of cleaning and sanitation of the
multipurpose plant production area is adapted for the
purposes foreseen of maintaining the area inside the
approaches of established acceptance for this type of
plant.
Disinfectants hat are used (Surfalyse to the 0.25%
(Aldehydes), Bactylisine to the 0.25% (quaternary
Ammonium) for floors, walls and curtains in
combination with Etanol to the 70% they are troops to
the eight hours of carrying out the cleaning, some of
the points leave from the approach of acceptance
considering the execution of operations that they are
carried out in the adjacent zone to the same, taking
like recommendation proceed a cleaning more located
in these points
REFERENCES
21 CFR 211.160 (b), Laboratory Controls 9revisions as of a2
may 2006).
21 CFR 211.67, Equipment Cleaning and Maintenance
(revisions as of a2 may 2006).
Adner, Niklas, and Sofer, Gail. Biotechnology Product
Assessment, Part 3: Chromatography Cleaning
Assessment. April 1994, p. 44.
Annex 15 to the EU Guide to Good Manufacturing Practice:
Qualification and assessment, European Commission,
Brussels, July 2001.
CANADA Health Products and Food Branch Inspectorate,
Cleaning Assessment Guidelines, available at:
http://www.hc-sc.gc.ca/dhp-mps/compli-conform/gmp-
bpf/assessment/cleaning-nettoyage_e.html (09/09/03).
EC Guide to good manufacturing practice revision to annex
1, 2008.
FDA, Guide to Inspections of Cleaning Assessment, 1993.
Feuerhelm. D and Blank GS. Documentation: Its Not Just
Paper Work How to Plan, Organize, and Assemble Your
Process Assessment Documentation. BioPharm, pp. 18
21, August 2001,
J. Zayas, H. Coln, O. Garced and L.M. Ramos, J. Pharm.
Biomed. Anal. 41 (2006), pp. 589593.
Katona Z, Vincze L, Vgh Z, Trompler A, Ferenczi-Fodor K,
Cleaning assessment procedure eased by using
overpressured layer chromatography. J. Pharm. Biomed.
Anal. 22, pp. 349353, 2000.
Lakka, N.S. , Reddamoni, S.Y. Prasad, V. Sivakumar, K.
Cleaning validation method for residual estimation of
amc and rutin on surface of pharmaceutical
manufacturing equipment with swab sampling
technique by using HPLC UV method. International
Journal of Pharmacy and Pharmaceutical Sciences
Volume 6, Issue 1, Pages 409-414, 2014
M. Moser, G. Calderari and P. Morini, Chima 54 (2000), pp.
731733.
M.J. Nozal, J.L. Bernal, L. Toribio, M.T. Martin and F.J. Diez,
J. Pharm. Biomed. Anal. 30 (2002), pp. 285291.
P. Yang, K. Burson, D. Feder and F. Macdonald, Pharm.
Technol. 29 (2005), pp. 8494.
PIC/S Secretariat. Recommendations on validation master
plan installation and operational qualification non-sterile
process validation cleaning validation PI 006-3 25
September 2007
R. Klinkerberg, B. Streel and A. Ceccato, J. Pharm. Biomed.
Anal. 32 (2003), pp. 345352.
Regulacin 16-2012 Directrices sobre BPF de Productos
Farmacuticos del CECMED
Sartain Elaine.K. Designing a Cleanroom Disinfectant
Program to Meet Production Requirements and
Regulatory Expectations Controlled Environment,
Advances in Biomedical Engineering Research (ABER) Volume 2, 2014 www.seipub.org/aber
27
December 2004.
United States Pharmacopeia Forum USP <1116>
Microbiological evaluation of clean rooms and other
controlled environmets. 2012
Vellutato Jr. Developing compliant and effective cleaning
and disinfection methodologies in GMP controlled
environments. Cleanrooms august, 2006.
Wenzel Brenda.M. Assessment Training: How Do You Do
It? Wednesday, November 09, 2005 - Journal of GXP
Compliance, Volume 6 Number 2 January 2002