Qualification and Continuous Validation of A Compressed Air System Used in The Biotechnological Industry - IVT

5/20/2021 Qualification and Continuous Validation of a Compressed Air System Used in the Biotechnological Industry | IVT
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Qualification and Continuous Validation of a Compressed Air System Used
in the Biotechnological Industry
By Adonis Senra , Alberto Leyva , Gudelia Pérez , Iriac Bisquet , Manuel Montané , Rodolfo Valdés ,
Sonny Robles , Tania de la Cruz Dec 15, 2017 8:00 am EST
Abstract
Compressed air is widely used in the biotechnological industry. Impurities in compressed air may
jeopardize intermediate and nal products leading to quality deterioration. The quali cation and
validation of compressed air systems is mandatory to obtain a compressed gas with all quality
attributes. This study aims to show evidence in quali cation and continuous validation of the
compressed air system of a production plant that produces multiple active pharmaceutical
ingredients obtained by biotechnological processes such as: recombinant hepatitis B surface
antigen, human epidermal growth factor, granulocyte colony stimulating factor and p64K-r within
others. The results demonstrate that the total hydrocarbon content in all determinations were ≤ 0.1
mg/m3. In all analyzed samples, dew point temperature values were below 7 °C showing the
stability and quality of the distributed compressed air. Compliance to speci cations established by
ISO guide 8573-1:2010 for non-viable particles was also required. In addition, a microbial growth
level below 2 CFU/m3 was detected con rming the system reliability. All active pharmaceutical
ingredients ful lled the quality speci cations, which demonstrate the non-deterioration of the
products. In conclusion, the quali cation and maintenance of the validation status of the
compressed air system according to international standards ISO 8573.1:2010, about ensuring the
quality and purity, class of 1.5.2, are in agreement with current biopharmaceutical industry
requirements.
Introduction
Compressed air is one of the main ancillary services in pharmaceutical manufacturing facilities, as
well as a critical utility system for the operation of biotechnological plants in compliance with the
current Good Manufacturing Practices (cGMPs) [1]. In this regard, the ISO 8573-1:2010 is an
important international standard operation document that provides speci cations for a variety of
compressed air purity classes [2]. In the production plant, two ZR 90 VSD Atlas Copco compressors
were used to generate instrument and process air employed in di erent critical operations, such as
fermentation and nal ltration processes for manufacturing several recombinant proteins.
In the biotechnological industry, validation plays an essential role in producing high quality
pharmaceutical products and is a mandatory requirement demanded by regulatory authorities
worldwide to regulate the pharmaceutical and medical device production. Thus, it has to be
assumed that equipment, utilities, or facilities that are not validated might produce inferior or
unreliable outputs [3].
Compressed air systems require an adequate quali cation and validation. This is the process of
collecting and evaluating data to draw scienti c evidence that the system is capable of consistently
delivering quality products. Also, validation involves con rmation by examination and provision of
objective evidence that the particular requirements for a speci c intended use are ful lled [4, 5].
In that sense, a wide dissemination of compressed air qualities have been carried out [2, 6].
However, the validation results for a compressed air system as a quality issue has not been
reported yet.
Therefore, in this study, the quali cation and maintenance of the validation status of a compressed
air system in the production plant at the Center for Genetic Engineering and Biotechnology
according to international standard operation ISO 8573-1:2010 is reported as part of a robust
quality assurance plan for this critical utility, to ensure the quality and safety of the manufactured
products such as: the recombinant proteins as hepatitis B surface antigen (r-HBsAg), human
epidermal growth factor (hr-EGF), granulocyte colony stimulating factor (r-GCSF) and the
recombinant protein p64K-r .
Materials and methods

Compressed air system characteristics
The compressed air system provides service to meet the air for the process and instrumentation
needs in the production plant of the Center for Genetic Engineering and Biotechnology in Havana.
The system includes two screw oil-free compressors up to 14.7 m³/min capacity (Atlas Copco model
ZR 90 VSD), a 3 m3 storage tank followed by a 1 µm pre- lter for particles, two refrigeration air
dryers and a ltration unit comprised of a 0.01µm ne particle lter and activated carbon lter for
removing possible vapors of oil and other hydrocarbons. The system was designed to obtain quality
class 1:5:2 air according to ISO 8573-1:2010. The graphic representation of the system is shown in
Figure 1. For the validation of the compressed air system, the following sampling and distribution
https://www.ivtnetwork.com/article/qualification-and-continuous-validation-compressed-air-system-used-biotechnological-industry 1/7
points were selected. Point SA-PM005: System output, point SA-PM007: Process air output, point
SA-PI120: Final ltration area, SA-PI069: critical point.

Figure 1. Diagram of the compressed air system used to generate air for the production of r-HBsAg,
hr-EGF, r-GCSF and p64K-r.
Methodology
The continued process veri cation of compressed air systems was carried out taking into account
the information collected between 2014 and 2016. This data is continuously reviewed and analyzed
to determine whether or not the system being monitored is in a state of control according to the
established classes for quality attributes. Table 1 shows the mandatory requirements of the ISO
guide 8573-1:2010 as it is applied to particle, water content, oil and microbiology.
ISO 8573- Solid particulate Water Oil

1:2010
3
Class Maximum number of particles per m Mass concentration Vapor pressure Dew Liquid Total oil
point
0.1- 0.5 µm 0.5- 1 µm 1- 5 µm mg/m3 °C g/m3 mg/m3
0 As specified by the equipment user or supplier and more stringer than Class 1
1 ≤ 20 000 ≤ 400 ≤ 10 ≤ -70 ≤ 0.01
2 ≤ 400 000 ≤ 6000 ≤ 100 ≤ -40 ≤ 0.1
3 ≤ 90 000 ≤ 1000 ≤ -20 ≤1
4 ≤ 10 000 ≤ +3 ≤5
5 ≤ 100 000 ≤ +7
6 ≤5 ≤ +10
7 5 a 10 ≤ 0.5
8 0.5 a 5
9 5 a 10
X > 10 > 10 >5
Table 1. The ISO speci cations for compressed air, ISO8573.1-2010. The alert limit for microbiology
was established during the quali cation, start up and validation of the system with a values of 10
cfu/m3. Di erent purity classes (A.B.C) to the compressed air system are marked in yellow.
Microbiological control
The Pinocchio Super II, a microbial impact sampler was used to test the microbiological quality of
compressed air. A compressed air source was connected to the Pinocchio Super II system and the
ow meter regulated to achieve the required ow rate in order to obtain the required total sample
volume according to ISO 8573-7 [7]. One cubic meter in each sampling point was taken for
monitoring the system twice a month. Petri plates impacted were incubated at 20-25oC for 3 days,
followed incubation at 30-35oC for 2 days to promote the microbial growth in Tryptone Soya Agar
medium. Alert and action limits were established for this quality attribute.
Pressure dew point determination

Thermohygrometer Testo 635-2 with the precision pressure dew point (PDP) probe for
measurements in compressed air systems, including certi cate with test point -40°C, was used to
measure PDP in compressed air systems according to ISO 8573-3 and 9 [8, 9].
Oil determination
The determination of oil vapour content was carried out weekly according to ISO 8573-2 [10] using
the Aerotest simultan HP. Dräger tubes were used for the determination of oil concentrations in
pressurized gases, with a detection limit of ≤0.1 mg/m3 (class 2).
Solid particle counting

A laser particle counter (LPC) coupled to a high pressure di usor for sampling gases was used for
assessing the solid particles content in the compressed air system according to ISO 8573-4 [11].
One cubic meter in each sampling point was taken for monitoring the system twice a month.
Production of recombinant proteins

The r-HBsAg [12], hr-EGF [13], r-GCSF [14] and p64K-r [15] were manufactured at the Center for
Genetic Engineering and Biotechnology in Havana using the compressed air generated over a two
year testing period.
Results and discussion

Currently, even the Food and Drug Administration (FDA), with its high standards for pharmaceutical
and food industries, hardly gives any advice about the quality of the compressed air used in
production processes or methods, for ensuring and/or monitoring its quality. At industrial level, the
compressed air quality is de ned by the standard ISO 8573-1:2010. The analysis of the results was
carried out for each quality characteristic measured at the points indicated in the quality
documents.
Quali cation of the Compressed air system

The typical three-stage quali cation approach was followed, starting with the Installation
Quali cation (IQ), followed by the Operational Quali cation (OQ), and nally the Performance
Quali cation (PQ). In the rst step installation of all components and materials speci ed was
con rmed. In the OQ, all the operational functions of the equipmentwere tested for satisfactory,
applying an OQ protocol generated internally, which covered the whole system, according to
Thiesset JP [16]. Table 2 shows the quali cation results for each element of the system.
Element IQ OQ PQ
Air compressor yes yes no
Tank yes yes no
Air dryer yes yes no
Reducing air yes yes no
Filtration unit yes yes no
Compressed air system yes yes yes
Pipeline distribution yes yes yes
Table 2. Quali cation process for each system element
Performance Quali cation (PQ)

The purpose of the PQ was to demonstrate the system that produces and distributes compressed
air meets the ISO 8573-1:2010 requirements over a suitable time period. The quali cation period
was chosen to create balance between time and testing burden, demonstrate a robust system
(reliability), and provide assurance that the system would continue to be monitored in this manner
after completion of the quali cation testing.
Total hydrocarbons
In the pharmaceutical industry, oil-free compressed air quality is required because if oil substances
come into contact with the product being manufactured, it can a ect the product and cause
productivity losses. Nevertheless, having an oil-free compressor does not necessarily mean that
the compressed air is oil-free, as the term “oil-free compressor” refers only to the compression
chamber, not the compressor system as a whole, or the resulting compressed air quality. Several
contaminants in the compressed air generally can be attributed to the quality of the air drawn into
the compressor, the operation of the air compressor and compressed air storage devices as well as
the distribution system [17]. For this reason, it is necessary to carry out an adequate monitoring of
the oil vapor content as a way of the contamination of product or equipment to eliminate
substances that could reasonably expected to have an adverse e ect in the quality of the product
as it raises 21 CFR 820.70 (e) [18]. Hydrocarbon content in all measurements were carried out in the
PQ ≤ 0.1 mg/m3; of a total 202 measurements hydrocarbon content in 10 samples were found,
which represented the 4.95 % detected. However, the measured values in the points SA-PM005
and SA-PI069 are between of the speci cation limit for class 2 (Figure 2), which demonstrated that
the concentration of hydrocarbon ≤ 0.1 mg/m3 is not toxic for the cells during process fermentation
and subsequently the integrity of the nal product as reported Sikkema et. al in 1995 [19].
Therefore, this quality of air will be used in the processes and operations of the manufacturing
plant without toxicity risks.
Figure 2. Results of hydrocarbon content during two years of operating of the compressed air
system. The sampling frequency established in the monitoring plan was once per week for the
sampling points and point of use. The values on 0.1 mg/m3 were eliminated changing the ltration
unit. Speci cation limit represent the purity class to this parameter (class 2).
Pressure dew point

The suitable control of pressure dew point temperature is one of the most important aspects to
maintain the quality of air produced by compressed air systems in the pharmaceutical industry
[20]. Tthe sterility of the compressed air supplied to the fermenters is paramount to prevent
contamination that could lead to lower yields and product batch losses, but also the use of
refrigerant dryers to drive down the temperature of compressed air to 2-5 °C (36-41°F). It is also
important to prevent cell desiccation and product losses in the fermentation processes. It is in
contrast to those applied in the electronic industry, which uses very low temperature (-20 and -70
°C) as a requirement to maintain the electronic device quality [21]. As a result, Figure 3 illustrates
values of dew point temperature which were below 7 °C in all the analyzed samples. The lowest
value reported was 2 °C, which is quite distant to extremely dry values that can a ect the cell
integrity, and demonstrated the stability and quality of the distributed compressed air according to
class 5 as stated in ISO 8573-1:2010. On the other hand, these results eliminate the risks associated
with dew point levels out of control that may include a failure of the equipment by the
condensation in process lines avoiding the corrosion in the pipeline. In addition, the combination of
condensate water and hot compressed air provides the ideal environment for microbial growth and
subsequent pollution of the environment in the clean room as reported by Cowan in 2013 [20].
Figure 3. Results of pressure dew point during two years of operation of the compressed air
system. The sampling frequency established in the monitoring plan was once per week for the
sampling points and point of use. Speci cation limit represent the purity class to this parameter
(class 5).
Non-viable particles
Particulates found in compressed air come from the environment intake air (dirt, bacteria, etc.) and
the compressing system (compressor and piping distribution system) itself. One of the
requirements described in the ISO 8573-1:2010 for the compressed air quality is stated to the non-
viable particle content in the three micron size range allowed. For assessing this quality, a high
pressure di user coupled to LPC{spell this out to de ne it} was used. Compliance of the
speci cations established in ISO guide 8573-1 for non-viable particles was demonstrated. However,
in 0.1-0.5 µm range only the values between 0.3 to 0.5 µm can be determined due to counter
particle used. In this range, seven measurements were ≤10 number of particles per cubic meter.
Nevertheless, all results demonstrated the quality of compression process and distributed air to the
di erent areas and process operations (Figure 4). These results dshowed the e ciency and
integrity of ltration units as well as the quality of piping distribution, which avoid the
contamination of clean areas that use this service for di erent manufacturing processes. This
condition is in compliance with the FDA guidance for Sterile Drug Products Produced by Aseptic
Processing Current Good Manufacturing Practice [1].
Figure 4. Particle counts in 3 ranges according to ISO guide 8573-1:2010 to class 1 during two years
of operation of the compressed air system. (A) Range 0.3-0.5 µm. (B) Range 0.5-1 µm. (C) Range 1-5
µm. One cubic metric in each sampling point was taken for monitoring the system twice a month.
Speci cation limit represent the purity class to this parameter (class 1).
Microbiological analysis
Microbial content is not included as a purity requirement for compressed air system classi cation
in the Biotechnological industry. However, the authors point out that it is important to assess
bacterial contamination level in the compressed air, because it is used at di erent stages of the
manufacturing processes for numerous applications such as process gas supply, air to the
fermenter, instrument air to the operation of equipments and critical systems; and control gas with
open vent into the clean room [22, 23]. Taking into account, the microbial sampling was performed
as it is described in the Material and Methods section. As shown in Figure 5, all results (points SA-
PM005 and SA-PM007) were below the alert limit established for this quality attribute (10 CFU/m3).
The highest value was 2 CFU/m3, which did not a ect the quality of the compressed air in the
distribution lines and the clean room area, according to FDA Aseptic Filling Guidance document, EU
GMP Annex 1 2008 and WHO Environmental Monitoring of Clean Rooms [1, 24, 25]. No microbial
growth was detected in 86 samples as part of the total microbial count, which demonstrated that
86% of assessed sample results were 0 CFU/m3. In addition, these results corroborated the
sampling condition (e.g., control of the ow rate, pressure reduction, sampling time) and testing
requirements were performed as described ISO 8573-7 [7]. Therefore, although few regulatory
agencies such as FDA [1] refer slightly to the in uence of microbiological quality in the environment
when the gas is introduced, the authors recommend that this issue should be taken into
consideration due to its importance as a criterion of purity classi cation for compressed air
systems used mainly in the Biopharmaceutical industry.
Figure 5. Results of microbial content in the compressed air system during two years of operation.
The alert limit established in compressed air system for this quality attribute was 10 cfu/m3. In 100
determinations (twice a month per each point of use) only microbial growth in 14 samples was
found.
Application and in uence of compressed air in manufacturing of di erent recombinant

proteins
In the biotechnological industry, compressed air is used in a number of applications as a means of
aeration to fermentations and nal ltration process. In that sense, the control of di erent quality
attributes of compressed air used in direct contact with product is important. As shown in gure 4,
the values of PDP were ≤ 7°C, which complied with the speci cation to class 5 corresponding to
water content. The stability of this characteristic allowed to show no dilution by the elevated water
content (high dew point temperature) or dead cell culture by desiccation due to the low dew point
temperature values corresponding to class 1 and 2 used in electronic industry [26, 27]. Besides, the
control of this quality attribute allowed to obtain high expression level and purity in nal product
with respect to speci cation (Table 3). On the other hand, the nal ltration in all cases was suitable
where the sterility test of all lots (129) was satisfactory (Table 3). The supply of sterile air and a
suitable physical parameter to the fermenter and nal ltration prevented costly contamination,
while maintaining the conditions of the culture, and lowered the yields or even total product batch
loss of several recombinant proteins manufactured in the production plant.
Production Samples Fermentation Specified Expression SDS- Specification Sterility test

Performance Level PAGE purity (%) (%)
(%) (%)
r-HBsAg 14 3.5 > 1.5 97.9 >95 pass the test
p64K-r 10 26.5 >20 96.6 >96 pass the test
r-GCSF 17 26.3 >8 99.1 >97 pass the test
hr-EGF 88 65.9 >25 99.7 >98 pass the test
Table 3. Quality attribute of di erent recombinant proteins obtained in the production plant. In all
cases, results were satisfactory.
Conclusion
The results of this work demonstrate that the compressed air system of the production plant was
e cient in removing dirt, dust, oil vapor and microbial contamination. The quali cation and
validation of the system performed during two years to prove its reliability demonstrate the
robustness of the compressed air system for producing and distributing compressed air with the
required quality assurance. According to the international standard ISO 8573-1:2010 classes 1.5.2,
the compressed air system is in agreement with industry requirements.
Acknowledgments
The authors thank their colleagues from the Process Control Department and Microbiology
Laboratory for analytical support. They are also grateful to Prof. Elizabeth Díaz López and Sorange
Hernandez Fernandez for reviewing the English.
References
1. FDA (2004) Guidance for Industry Sterile Drug Products Produced by Aseptic Processing —
Current Good Manufacturing Practice, U.S. Department of Health and Human Services Food
and Drug Administration, Rockville, MD, USA
2. ISO 8573-1:2010 Compressed Air Contaminants and Purity Classes, International Standards
Organization, Geneva, Switzerland
3. J. Agalloco and F. J. Carleton, Validation of Pharmaceutical Process, 3rd Edition, USA, New
York: Inform Healthcare, 2008.
4. EudraLex - Volume 4 Good Manufacturing Practice (GMP) Guidelines, Annex 15 Quali cation
and Validation, 2015, March
5. FDA (2011) Guidance for Industry Process Validation: General Principles and Practices, U.S.
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Standards Organization, Geneva, Switzerland
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Geneva, Switzerland
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Organization, Geneva, Switzerland.
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5/20/2021 Qualification and Continuous Validation of a Compressed Air System Used in the Biotechnological Industry | IVT

ABOUT IVT MEMBERSHIP JOURNALS PRODUCTS EVENTS WHAT'S NEW R

Sonny Robles , Tania de la Cruz Dec 15, 2017 8:00 am EST

Materials and methods

ISO 8573- Solid particulate Water Oil

0.1- 0.5 µm 0.5- 1 µm 1- 5 µm mg/m3 °C g/m3 mg/m3

1 ≤ 20 000 ≤ 400 ≤ 10 ≤ -70 ≤ 0.01

2 ≤ 400 000 ≤ 6000 ≤ 100 ≤ -40 ≤ 0.1

3 ≤ 90 000 ≤ 1000 ≤ -20 ≤1

X > 10 > 10 >5

Pressure dew point determination

Solid particle counting

Production of recombinant proteins

Results and discussion

Quali cation of the Compressed air system

Air compressor yes yes no

Tank yes yes no

Air dryer yes yes no

Reducing air yes yes no

Filtration unit yes yes no

Compressed air system yes yes yes

Pipeline distribution yes yes yes

Table 2. Quali cation process for each system element

Performance Quali cation (PQ)

Pressure dew point

Application and in uence of compressed air in manufacturing of di erent recombinant

Production Samples Fermentation Specified Expression SDS- Specification Sterility test

r-HBsAg 14 3.5 > 1.5 97.9 >95 pass the test

p64K-r 10 26.5 >20 96.6 >96 pass the test

r-GCSF 17 26.3 >8 99.1 >97 pass the test

hr-EGF 88 65.9 >25 99.7 >98 pass the test

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