Design And Validation Of Cleanrooms For Interferon-Alpha Manufacturing

Background

Interferons are probably the first cytokines to be discovered. During the 1950s, scientists observed various animal cells when exposed to the virus, they automatically become resistant to attack by other viruses(Hanaoka and Takeuchi, 2013). This resistance was induced by interferons secreted by virally infected cells.  Interferons are produced by different cell types of which they exhibit their biological effects in various ways which include induction of cellular resistance to viral attack, regulation of growth and differentiation of various types of cells, control of the most aspects of the immune system and sustenance of early stages of pregnancy in animal species(Conner et al., 2014). In human, there are three known categories of interferons namely INF-alpha, INF-b, and INF-y. The interferon –alpha produces multiple interferons when well purified. In human beings, there are about 24 genes that exist for the production of about 16 well-known interferon alphas(Hanaoka and Takeuchi, 2013). These interferons are normally classified into two classes namely class I and II where class I contain 15 interferon- alphas and type II contains only one interferon alpha(Bax et al., 2013).

Like many cytokines, interferon alpha is produced as a drug using biotechnology techniques. The most commonly produced is interferon- alpha -2b(Hanaoka and Takeuchi, 2013). The drug is used in the treatment of hepatitis B and C, malignant melanoma, hairy cell leukemia, and Aids-related the Kaposi Sarcoma(Bax et al., 2013). Like many vaccines and biotech produced drugs, the interferon alpha drug is produced under a well-controlled cleanroom due to its fragility nature(Ramana, Xavier and Sharma, 2017).

 A cleanroom is a controlled placement where various biotechnological and medicinal products are manufactured(Dixon Anne Marie, 2007). All cleanrooms are not similar in terms of structure and size but apply similar standards. According to ISO 14644, the concentration of airborne particles in a controlled room is specified to certain standard limits depending on the product manufactured(ISO, 2015). Most of the contaminations in a cleanroom are generally from workers, equipment used and from the facility itself(Zhang, Sun and Ma, 2017). The purification of any drug manufactured in a cleanroom depends on the design used and monitoring processes(Whyte, Lenegan and Eaton, 2016). This study seeks to demonstrate classifications of cleanrooms, various impurities that can be present in a certain cleanroom, the design of a clean room with a complete schematic relevant to interferon –alpha and testing and monitoring required in a cleanroom evaluation.

There are various causes of contamination in a cleanroom which include employees and employers, equipment, processes, and environmental surroundings(White, 2009). In a cleanroom for interferon-alpha manufacturing, bacteria are the most important contaminants(Keller, 2012). Almost all the contaminants occur from workers in the cleanroom thus the number needs to be regulated. This is due to the fact that, a larger number of people in a cleanroom will have a direct effect on the quantity and quality of air thus affecting the number of particles available(Gommel, Kreck and Holzapfel, 2014). In addition, the efficiency and air ventilation in a clean room are normally affected by the type of clothing and equipment used by the workers.  The more effective the clothing used in terms of preventing dispersion, the less the air in the clothing fabrics thus ventilation is enhanced.

Sources of Contamination in Cleanrooms

Another source of contaminant in a cleanroom is the equipment used(Keller, 2012). The most significant objective at this point is by removing the particles from the equipment before limitations are made for removing dust particles once the worker has entered the cleanroom(Gommel, Kreck and Holzapfel, 2014). In addition to equipment, the airborne contamination coming from outside is a normal problem when it comes to cleanrooms. This happens when the cleanroom is poorly designed with a lot of ventilation not well sealed(Whyte, Lenegan and Eaton, 2016). Other than that, contamination can be introduced to the cleanrooms when materials are poorly distributed through poorly designed changing areas and airlocks(White, 2009). This can contribute to either air or surface contamination.

Pharmaceutical drugs production requires special requirements in order to reduce the risks of contaminations from micro-organisms. Implementation of those regulations and standards requires personal attitude to work, training and operational experience(Ranade, 2010). Sterilized medications like interferon alpha-2b must be produced in the organized cleanrooms normally referred to as clean zones(World Health Organization, 2011). The clean zones must be equipped with personnel, materials, and equipment. Due to that fact, cleanrooms must support purity levels at all standards. There should be enough air supply with necessary efficiency filters(WHO, 2016a).  Cleanrooms for manufacturing of interferon alpha-2b must be classified according to the environmental requirement in order to reduce chances of product contamination(Hiyama, 2010). Filling, product preparation and the original materials must be done in separate cleanrooms. Every working operation need a certain purity level of environmental conditions.

Classification of cleanrooms is done in accordance with the cleanliness of the air. Normally, there are various general standards that are used in designing cleanrooms.  In the United States, the Federal Standard 209 also known as ‘ Clean Room and Work Station Requirements, Controlled Environments’ developed in the 1960s  has been used in designing of cleanrooms(ISO, 2015). The standard determines the classification of cleanrooms by measuring particles more than 0.5 um.  The ISO( International Organisation for standards)- 14644-1 also known as ‘Cleanrooms and Associated Controlled Environments’ is the most common standard used by both the United States and European Union in the classification of cleanrooms(ISO, 2015). Below is a table that demonstrates ISO-14644-1 classification as compared to Federal Standards 209.

                                                     Table 1:A table of ISO 14644-1 classification as compare to Federal  Standards 209

Class	Maximum particles / m3	Federal Standards 209 equivalent
>= 0.1um	>=0.2um	>=0.3um	>=0.5um	>=1um	>=5um
ISO 1	10	2.37	1.02	0.35	0.083	0.0029
ISO 2	100	23.7	10.2	3.5	0.83	0.029
ISO 3	1000	237	102	35	8.3	0.29	Class 1
ISO 4	10,000	2,370	1,020	352	83	2.9	Class 10
ISO 5	100,000	23,700	10,200	3,520	832	29	Class 100
ISO 6	1.0 * 106	237,000	102,000	35,200	8,320	290	Class 1000
ISO 7	1.0 *107	2.37 * 106	1,020,000	352,000	83,200	2,900	Class 10000
ISO 8	10 * 10 8	2.37 * 107	1.02 *107	3,520,000	832,200	29,000	Class 100, 000
ISO 9	10 * 10 9	2.37 * 108	1.02 * 108	35,200,000	8,320,000	290,000	Room air

Requirements for Cleanrooms for Interferon-Alpha Manufacturing

                                                                                                     (ISO, 2015)

The above table represents the ISO 14644-1 that specifies the classification of air cleanliness taking consideration of the airborne particles in clean zones and cleanrooms. In addition the revised ISO 14644-1, 2015 have added more separative devices and specifications.  As shown in the table, the ISO 14644-1 only shows particles in terms of the distribution based on the lower limit only that is between 0.1um and 5um(ISO, 2015).  Airborne particles concertation is usually determined by the light scattering airborne particles counter (LSAPC) which is normally equal to or greater than specifically desired particles at given sampling locations(ISO, 2015). The standard classification does not provide classifications of particles that are outside the threshold. In addition, the ISO -14644-1 cannot be used to classify chemical, radiological or physical particles.

In 2015, the ISO 14644-1 and 2 were revised and several changes of classification have been introduced. The ISO 14644 consist of ten parts which include classification of cleanliness by particle concentration,  monitoring of cleanroom performance related to cleanliness by particle concentration, the test methods, design and construction of the cleanrooms, operations, separate devices, classification of air cleanliness by particle concentration and classification of surface cleanliness by chemical concentration(ISO, 2015).  However, the first classification is the most commonly used for biotechnology and pharmaceutical cleanrooms.  One of the major revision that was done on ISO 14644-1 in 2015 is a simplification of the classification process in order to evaluate 95% of the upper confidence limit (UCL) for lower sample location number(Nomen, 2015). This was done in order to update the standards to the current industrial requirements and reasoning(ISO, 2015) . Other than that, the review of the classification was made to make the cleanroom applications more applicable. For instance, airborne contamination is not expected to be evenly distributed. Furthermore, the current classification has avoided any possible changes for ISO classes 1-9.

For manufacturing of interferon alpha-2b, four local zones are needed namely A, B, C and D(Nomen, 2015).  An  A-local zone is used for operations that can accommodate high risks for interferon-alpha drugs quality. These include activities like bottle opening, filling, and closing of bottles(Gommel, Kreck and Holzapfel, 2014). In a normal cleanroom, such a zone requires laminar airflow that provides verbosity equivalent to 0.54m/s. The B-zone requires high levels of acetic techniques and it is usually for preparation and fulfills. The final two zones are only used for manufacturing interferon alpha drugs only where minimal or no contamination is expected(World Health Organization, 2011).  World Health Organisation (WHO) has provided various grades of the maximum permitted particles for the production of vaccines and pharmaceutical products(WHO, 2016b). Given that, interferon alpha has been used to generate various vaccines, the following table from WHO demonstrate the required cleanroom maximum permitted airborne particle concentration per air grade.

Classification of Cleanrooms

                                                  Table 2: A table demonstrating standard cleanrooms for interferon-alpha production

Grade	At rest The maximum permitted particles/m3	In operation The maximum permitted particles/m3
	>=0.5um	>=0.5um	>=0.5um	>=0.5um
A	3,520	20	3,520	20
B	3,520	29	352,000	2,900
C	352,000	2,900	3,520,000	29,000
D	3,520,000	29,000	Not defined	Not defined

                                                                               (World Health Organization, 2011)

The above Grades correspond to the Federal Standards 209 and ISO 14644-1 classifications standards. WHO recommends that companies manufacturing interferon alpha to follow the above grades for quality assurance. However, a company may choose different classification other than that recommended by WHO but it should present a table of own classification that goes hand in hand with the international standards. During cleanroom preparation, the particles sizes between 0.5um and 5um should be measured(Brás and Rohr, 2013). Viable microorganisms can be calculated by measuring both passive and active air sampling of workers and surfaces(Brás and Rohr, 2013). As demonstrated by the table above, the measurement should be considered in both dynamic and static conditions for microorganisms and particles(Whyte, Green and Whyte, 2012). For instance, in B, C, and D grades the number of particles should be measured in accordance with the size of the room, equipment used and workers available or working in a cleanroom. Appropriate filters such as HEPA should be applied in grade A, B and C. A clean-up period of about 30 minutes should be provided at rest(Meshram et al., 2017). In addition, adequate alerts and operation limits should be set for particulate and microbial monitoring(Channaiah, 2016).  Other parameters such as relative humidity and temperature have no connection in the purification process and thus they should depend on the nature of manufacturing.

The interferon alpha drugs are manufactured using the microbial upstream operations. This is done by the use of Escherichia choli. This process involves the growth of Escherichia choli that has been programmed using human genes to produce interferon alpha drugs(Nomen, 2015). The production of substances begins by throwing the microbial cells into the bank(Hanaoka and Takeuchi, 2013).  The cell bank is normally designed to support the growth with adequate temperature for culture. The culture is then incubated in such a way it will allow an optimum environment for the production of the fermenters(Bax et al., 2013). The produced parameter is designed in such a way it can control all the desired parameters including the airflow, temperature, and pH. The combination of all these controls allows the Escherichia coli bacteria to have an optimal environment for growth. This process of fermentation normally uses the fed-batch process in order to allow high growth of microorganisms through feeding of additional supplements and specialized medium.

ISO-14644-1 Overview

After the fermentation process is completed, all microorganisms are harvested using the centrifuge approximately after 48hours. This is due to the fact that, the targeted interferon alpha is usually inside the Escherichia coli. However, the microorganism must be disputed to remove the targeted interferon alpha. This is usually done using the high-pressure homogenizer process. After the removal of interferon alpha, the cell debris must be removed using the centrifugation. The isolated inclusion bodies can be kept frozen before the further downstream process. Below is a picture that demonstrates the downstream process that is usually used in the production of interferon alpha.

                                               

                                            Figure 1: A diagram showing the downstream process of interferon-alpha production

Designing a cleanroom for manufacturing of interferon alpha as described above requires certain considerations(Meshram et al., 2017). These include operational efficiency, operational safety, product protection from contamination, facility protection and monitoring and maintenance(Whyte, Lenegan and Eaton, 2016).  Other than that, the whole manufacturing plant must be designed in such a way there are proper equipment arrangements, the material used is flowing in a considerate way and the personal flow is the way that reduces contamination(White, 2009). The final product flow should be well designed to prevent a mix-up with the waste flow(Abramowitz, 2016). Other than that, the firm should also have various segregation cleanrooms, adjacencies, expandability, and flexibility. Below is a schematic diagram that shows a typical manufacturing design of interferon alpha.                             

                                           Fig 2; A schematic picture of a single product with moderate segregation( interferon alpha)

As indicated in figure 2 above, the standard layout consideration for interferon-alpha production should have adequate adjacency as compared to the space available(Salaman-Byron, 2018). Other than that, a layout should consider the logical and good flow of equipment, personnel materials, and products with distinct cleanrooms(Sandle et al., 2014). A well-designed layout should also consider available of dirty equipment, and personnel passing through the same block or corridor(Sakraida, 2008). Airlocks should always be put into place as they are used as major separation points and maintenance of optimum pressure. The most cleanroom spaces should be located in the middle of the layout and surrounded by areas of lower classification as indicated in table 2.

Cleanroom Design and Validation for Interferon-Alpha Manufacturing

The most important aspect when designing a cleanroom for manufacturing of interferon alpha is air filtration.  High Efficacy Particulate Air(HEPA) filters should be put into place to allow the proper directional flow of air and control the pressures relationships between and within the adjacent spaces. Additionally, humidification and dehumidification should be controlled to maintain relative humidity plus the cooling and heating to maintain the desired optimal temperatures. The lowest particulate count is usually classified to cleanest rooms and it is usually achieved by use of HEPA filters for recirculation of rooms through the recirculation loop(Gommel, Kreck and Holzapfel, 2014). The more the clean a room needs to be the higher the rate of circulation is required. The degree of circulation can be measured by calculating the number of room air exchanges per hour(Nomen, 2015).  For instance, a number of exchanges that are required in the interferon alpha production cleanrooms are 300-480 changes per hour in the grade A rooms, between 60 and 90 in the grade B rooms and 20-40changes per hour in grade c rooms(Conner et al., 2014). However, the actual number of changes per hour depends on the personnel present, the level of activities and the level of contaminants thus they are dynamic in different firms.

Generally, the cleanrooms with grade A should be designed in such a way they have positive pressure as compared to other rooms. The basic use of airlocks is to separate the cleanrooms from colliders and the adjacent rooms(Xu, 2008). Comparing to the process rooms, they gowning rooms and airlocks should have negative pressures.  The only exceptions that can make are when live virus is being produced(Gommel, Kreck and Holzapfel, 2014). In such a scenario, the process rooms will have negative pressures and the airlock positive pressures. Most commonly used pressure differential between the clean rooms and the adjacent areas is between 15-20Pa. However, it is good to note that, when the doors open, the difference in pressure drop to zero(World Health Organization, 2011). This is the reason why airlocks should be installed in the connection points where the two doors should be used. The two doors in the airlocks should not be opened simultaneously and in most cases, the interlocking electricity controls between the airlock doors should be used(Hiyama, 2010). All rooms in a layout should be tightly sealed to enable all the pressures between rooms are maintained.

In most biotech drugs manufacturing designs, the companies use fixed processing piping and fixed stainless steel equipment. However, hose pipes can be used together with steel pipes to reduce the cost of production.  In addition, smaller plants can use disposable fermentation bags, equipment, storage bags, and filters. This enhances the reduction of investment cost, operating cost and increase flexibility for future expansion.

Conclusion

During the manufacturing process of interferon alpha, aseptic techniques must be observed by all the personnel to minimize contamination(WHO, 2016a). A gowning room must be created at grade D where individuals should change gowns when entering the process rooms. Gowns should have fewer cotton fabrics and regularly be sterilized to minimize microbial infections and particulates(Whyte, Lenegan and Eaton, 2016). All peoples components including vials, products, and stoppers should be assembled in a high-quality environment and also be sterilized. Only the small quantity of the final product should be tested to confirm therapeutic and sterility value(Fisher et al., 2018). The rest of the final product should only be indicated how they were processes and be confirmed it is safe for human use.  Examples of containers that are used for interferon alpha products include the vials, ampules for sterile water and syringes.  

The process of vials filling includes checking the vial weight, filling the desired product volume, inserting the vials stoppers and over sealing to secure the stoppers.  All vials must undergo a process of inspection including both manual or automatic inspections(Chen, Chen and Wang, 2014). This might be done in the filling process where rejected vials are discarded.  The ISO 5 environments are where the most aseptic processing should take place. This is due to the fact that, this environment where product and other parts are exposed(Sandle, 2014). The other classes of the standard are used in other environments depending on the potential impact the process will bring to the final product. During the filling process, dirty personnel and waste must be separated from clean operators and the products. This requires separate airlocks and corridors for clean and dirty activities(Abramowitz, 2016). Due to the presence of many dirty operators, it is recommended to use isolators during the filling process.

According to WHO, there are four major tests that are used in monitoring the microorganisms in a cleanroom which include settle plates, volumetric testing, finger labs, and contact plates(World Health Organization, 2011). However, not all test are done for every activity of monitoring a cleanroom. For class A, finger labs, settle plates, and volumetric testing must be done for every operation(WHO, 2016b). Depending on the plant, several samples can be taken for a test for every monitoring shift. The environmental monitoring should be performed according to the risks observed by the producer. Selection of a sample site must be considered when monitoring(WHO Expert Committee on Specifications for Pharmaceutical Preparations, 2006). Sample sites should be don inside the cleanroom where most individuals come into contact with the product(Conner et al., 2014). All clean rooms should be analyzed through the use of a risk assessment before the testing samples are taken(Zhang, Sun and Ma, 2017). Highly used areas should be monitored frequently as compared to low-risk areas. The risk-benefit relationship should be used to govern procedures and equipment monitoring accessibility high-risk zones(Abramowitz, 2016). The sample should be taken in a way that reduces more risks in high risks areas other than bringing sampling devices than increase contamination(Sandle, 2014). Where monitoring is considered unacceptable, the proof must be provided with proper documentation.

References

In the case of interferon alpha, a growth media and Escherichia Coli are used. Those microorganisms are settled for fermentation where supplements are usually added to promote growth(Channaiah, 2016). Growth monitoring testing is crucial when it comes to biotech drugs since, in the clean rooms where the processing take place, the growth media can develop into contaminants. Each company producing such drugs should form a set of program that allows growth media testing(Kumar and Ajeya Jha, 2015).  This includes waste testing and environmental monitoring. In environmental monitoring, the Agar plates are commonly used. The growth monitoring capacity of the growth media should be frequently be monitored using the Agar plates in every cycle(Eaton, Davenport and Whyte, 2012).  Using a standard time of incubation, the plates should be tested their ability to produce a low number of standard bacteria when cultivated. In addition, the time for recovery of such an organism should be monitored as they can affect the results of the product in the cleanroom(Xu, 2008).  In addition, fungi and bacteria used in the media should be tested if they have come resistance or not. The testing should be considered using the drug estimated period of expiry. The plates should be put in areas with high temperatures, airflow, turbulence, and low humidity in order to dry out or change their features(Zhang, 2015).  This is intended to kill the previously used bacterial and capture the new ones(Commissioner, 2016). After that, validation studies should be done in order to know which bacteria did not die under a specific condition and to determine how long the media can survive.

Volumetric air can determine the number of bacteria surrounding the product. Although active sampling can be used to identify the specific bacteria in the air, it is not usually recommended as a determinant of active contamination microbes(The Automotive Research Association of India, 2010).  The sample location of microbes can be chosen by the producer based on risk analysis. Risk assessment should be always the determinant of regular validation. However, a sampling of air microbes can be used as a determinant only if they pose a threat to the process of production(Klykens et al., 2013). The need for air microbes sample for the contamination determination and sensitivity should be determined by sample duration which always takes a lot of time. In addition, the air microbes can be monitored if they set a threat to aseptic process used. The recommended WHO sample size that should be taken is above one-metre cubic. However, where different microbial have been detected the sample size can be reduced depending on the manufacturer(World Health Organization, 2011). The design that should be employed to monitor and validate volumetric air should include the existence of important microbes and isolates plus the microorganism that are likely to affect the operators(WHO, 2016b).  A validation study should be conducted to determine the effect of delaying plates during transportation to the laboratory. This is usually done by setting a time limit that will dictate whether the microbes are viable or not during transport.  In addition, the manufacturer should assess the risk involve when transporting the microbes to the laboratory from the processing unit.

Settle plates are usually used to monitor the bacteria descending from the air over a plate. Although, the detection of settle plates depends on the type of microbe and their settling rates, settle plates can be assumed as the only method that can be used for continuous monitoring of microorganism in the cleanroom(WHO, 2016b).  The high-risk zones of contamination are considered places to place the settle plates. Settle plates should be placed in the process room but caution should be taken to avoid obstruction of the process or contamination of them(World Health Organization, 2011). The measurements from settle plates should only be taken when the levels of activities are high or durion the operation period. If the settle plates start drying, the new ones should be put into place to allow the maximum time is taken(WHO, 2016b). Validation data should always determine individual settle plates but not as a group. The manufacturer should set time to make sure the required data is obtained with certain shifts.

The process areas and other adjacent areas should be monitored using the above methods in a given period of time. If the company is not working for a certain period no monitoring should be put into place. Below is a table that demonstrates the recommended frequencies for monitoring in different grades.

                                                                         Table 3: different frequencies for monitoring in various grades 

Classification	Volumetric(2)	Settle plate(2)	Contact plate	Glove print
Grade A (filling operations)	Once per shift	Once per shift	Once per shift	Once per shift
Grade B	Daily	Daily	Daily	Daily
Grade C	Weekly	Weekly	Weekly	N/A
Grade D	Monthly	Monthly	N/A	N/A
UDAF in B	Once per shift	Once per shift	Once per shift	Once per shift
UDAF in C	Weekly	Weekly	Weekly	Weekly
UDAF in D	Monthly	Monthly	Monthly	N/A

                                                                                   (World Health Organization, 2011)

Conclusion

 Most drugs that are genetically engineered are produced through the use of biotechnology. Interferon alpha is among the biotech drug where the most commonly produced drug is interferon alpha 2-b. The drug is used for the treatment of various diseases and sometimes as a vaccine. These diseases include hepatitis B and C, malignant melanoma, hairy cell leukemia, and Aids-related the Kaposi Sarcoma. Considering many pharmaceutical and biotech drugs are produced under a well-controlled cleanroom, interferon alpha drug is not an exception. A cleanroom is considered a placement with highly controlled measures where various biotechnological and medicinal products are manufactured. For a cleanroom to be fully functional various international standards have to be taken into action. According to ISO 14644, the concentration of airborne particles in a controlled room is specified to certain standard limits depending on the product manufactured.  A cleanroom can have various contaminations which include personals, equipment used,  nature of production and from the facility itself. Classification of cleanrooms depends on the standards set international although some countries have their own standards. The commonly used standards include the ISO 14644-1 and the Federal Standards 209 of the US.  The ISO 14644-1 standards are used internationally especially by the European Union and the U.S. For the purposes of production of sterile medicines the WHO have provided a various classification of a cleanroom obtained from the above two major standards where the interferon alpha applies. When designing a layout for the manufacturing of interferon alpha there are various general conditions that are required to be met which include operational efficiency, operational safety, product protection from contamination, facility protection and monitoring and maintenance. In addition to that, the whole manufacturing company must be designed in such a way there are proper equipment arrangements, the material used is flowing in a considerate way and the personal flow is the way that reduces contamination. Monitoring of cleanrooms is crucial in the production of interferon alpha. In most cases, the monitoring targets the microorganism that can develop or be available in the cleanrooms. Some of the most common monitoring methods for microbes include settle plates, volumetric testing, finger labs, and contact plates. Other parameters like temperature, humidity, and pH also need to be monitored.

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